Synthetic antisense oligodeoxynucleotides targeted to human ache

ABSTRACT

A synthetic nuclease resistant antisense oligodeoxynucleotide (AS-ODN) capable of selectively modulating human acetylcholinesterase (AChE) production and a composition comprising at least one AS-ODN as an active ingredient. A nuclease resistant antisense targeted against the splice junction in the AChE mRNA post-splice message is disclosed. The synthetic nuclease resistant AS-ODNs are capable of selectively modulating human AChE production in the central nervous system or capable of selectively reducing human AChE deposition of the neuromuscular junction. The present invention also provides a method to restore balanced cholinergic signalling in the brain and spinal cord or reduce AChE in the neuromuscular junction in patients in need of such treatment by administering to a patient in need of such treatment a therapeutically effective amount of at least one of the synthetic nuclease resistant AS-ODN capable of selectively modulating human AChE production.

GOVERNMENT SUPPORT

Research in this application was supported in part by U.S. Department ofthe Army Contract DAMD17-86-C-6010. The U.S. Government has anonexclusive, nontransferable, irrevocable paid-up license to practiceor have practiced this invention for or on its behalf as provided by theterms of Contract DAMD17-86-C-6010 awarded by the U.S. Department of theArmy.

CROSSREFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. Ser. No. 08/850,347filed May 2, 1997 which is a Continuation-In-Part of U.S. Ser. No.08/318,826, filed Dec. 1, 1994, now U.S. Pat. No. 5,891,725, which isthe National Phase of PCT/EP93/00911 international filing date of Apr.15, 1993, priority Apr. 15, 1992 and U.S. Provisional Application No.60/035,266 filed Dec. 12, 1996; Provisional Application No. 60/037,777filed Feb. 13, 1997; Provisional Application No. 60/053,334 filed Jul.21, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of this invention is antisense oligodeoxynucleotides andpharmaceuticals based on them.

2. Description of Related Art

The ACHE gene encoding the acetylcholine hydrolyzing enzymeacetylcholinesterase (AChE, EC 3.1.1.7) is expressed in muscle, nerve,hematopoietic cells, embryonic tissue and germ cells. ACHE maps tochromosome 7q22 and encodes the primary enzyme, acetylcholinesterase(AChE, E.C. 3.1.1.7), which terminates neurotransmission at synapses andneuromuscular junctions.

The text Human Cholinesterases and Anticholinesterases by Soreq andZakut (Academic Press, Inc., 1993) provides a summation of thebiochemical and biological background as well as the molecular biologyof human cholinesterase genes. The text in its entirety is incorporatedherein by reference. Further, the text Transgenic Xenopus by Seidman andSoreq (Humana Press, 1996) provides a summation of the development ofthe Xenopus transgenic animal model. The text in its entirety isincorporated herein by reference. Articles by Beeri et al, 1995; Karpelet al, 1996; and the review articles by Lev-Lehman et al, 1997 andGrifman et al, 1995, 1997 provide further information on the developmentof antisense ACHE oligomers, the parameters for choosing sequences andtesting for efficacy and are incorporated herein by reference.

Briefly, AChE includes the peptide motif S/T-P-X-Z, which makes it apotential substrate for phosphorylation by cdc2 kinases, the generalcontrollers of the cell cycle. Most other substrates of cdc2 kinasesperform biological functions necessary for cell cycle-related processes.Thus, interference with either CHE or cdc2 transcription processes maybe expected to divert and/or arrest cell division, and controlling theseprocesses can be useful for several, medically important procedures.

Biochemical and histochemical analyses indicate that AChE is expressed,in high levels, in various fetal tissues of multiple eukaryotic specieswhere cholinesterases (ChEs) are coordinately regulated with respect tocell proliferation and differentiation. The specific role to beattributed to ChEs in embryonic development may hence be related withcell division, so that their biological function(s) in these tissues aretentatively implicated in the control of organogenesis.

In addition to its presence in the membranes of mature erythrocytes,AChE is also intensively produced in developing blood cells in vivo andin vitro and its activity serves as an acceptable marker for developingmouse megakaryocytes. Furthermore, administration of acetylcholineanalogues as well as cholinesterase inhibitors has been shown to inducemegakaryocytopoiesis and increased platelet counts in the mouse,implicating this enzyme in the commitment and development of thesehematopoietic cells.

A major hydrophilic form of AChE with the potential to be "tailed" bynon-catalytic subunits is expressed in central nervous system and musclewhereas a hydrophobic, phosphoinositide (PI)-linked form of the enzymeis found in erythrocytes. Alternative exons encoding the C-terminalpeptide in AChE were shown to provide the molecular origins for theamphiphilic (PI)-linked and the hydrophilic "tailed" form of AChE inTorpedo electric organ. The existence of corresponding alternative exonsand homologous enzyme forms in mammals suggested that a similarmechanism may provide for the molecular polymorphism of human AChE.cDNAs reported to date from mammalian brain and muscle encode thehydrophilic AChE form.

More specifically, three alternative AChE-encoding mRNAs have beendescribed in mammals (FIG. 11). The dominant central nervous system andmuscle AChE (AChE-T) found in the neuromuscular junction (NMJ) isencoded by a mRNA carrying exon E1 and the invariant coding exons E2,E3, and E4 spliced to alternative exon E6 [Li et al., 1991; BenAziz-Aloya et al., 1993]. AChEmRNA bearing exons E1-4 and alternativeexon E5 encodes the glycolipid phosphatidylinositol (GPT)-linked form ofAChE characteristic of vertebrate erythrocytes (AChE-H;) [Li et al.,1993; Legay et al., 1993a]. An additional readthrough mRNA speciesretaining the intronic sequence I4 located immediately 3' to exon E4 wasreported in rodent bone marrow and erythroleukemic cells [Li et al.,1993; Legay et al., 1993a] and in various tumor cells lines of humanorigin [Karpel et al., 1994].

AChE is the major cholinesterase (ChE) in nervous system cells. Sinceacetylcholine is produced mostly in the CNS, changes in AChE should becoupled to mental state.

The cholinergic theory of Alzheimer's disease [Coyle, et al, 1983;Slotkin et al., 1994] suggests that the selective loss of cholinergicneurons in Alzheimer's disease results in a relative deficit ofacetylcholine in specific regions of the brain that mediate learning andmemory functions and require acetylcholine to do so. The primaryapproach to treating Alzheimer's disease has therefore aimed to augmentthe cholinergic system. Reduced levels of acetylcholine in the brains ofAlzheimer's patients leaves a relative excess of acetylcholinesterase,the enzyme responsible for terminating nerve impulses during normalbrain activity by disposing of used acetylcholine (Soreq and Zakut,1993). A relative excess of acetylcholinesterase accentuates the growingcholinergic deficit by further reducing the availability ofacetylcholine. The most successful strategy to date for reinforcingcholinergic neurotransmission in Alzheimer's patients is pharmacologicalinhibition of acetylcholinesterase. Indeed, the only currently approveddrugs for Alzheimer's disease are potent acetylcholinesterase inhibitors(Winker, 1994), i.e. drugs that suppress the catalytic activity of theacetylcholine hydrolyzing enzyme acetylcholinesterase (acetylcholineacetyl hydrolase, EC 3.1.1.7, AChE) [Knapp et al., 1994]. This providesaugmentation of cholinergic neurotransmission, which is impaired in suchpatients due to the selective loss of cholinergic neurons. However, suchinhibitors do not reduce the amount of AChE protein, and there arerecent reports of actions of AChE, unrelated to its catalytic activity,in process extension [Small et al., 1995, Layer et al.; 1995, Jones etal., 1995; Darboux et al., 1996; Sternfeld et al., 1997] and amyloidfibril formation [Inestrosa et al., 1996].

Tacrine, the first and most well-characterized acetylcholinesteraseinhibitor used for treating Alzheimer's disease offers limitedpalliative relief to 30-50% of mild-moderately affected Alzheimer'spatients for up to 6 months [Winker, 1994; Krapp et al., 1994]. Thepositive, albeit partial, success of Tacrine attests to the utility ofthe cholinergic theory and the potential value of improvedanticholinesterase treatment for Alzheimer's disease. Another approveddrug, E-2020 (Aricept) has been reported to follow the same mode ofaction as Tacrine but at lower doses [Rogers et al., 1996]. A number ofother compounds are under development for inhibition ofacetylcholinesterase [Johansson and Nordberg, 1993]. All of these areaimed at blocking the fully folded protein from degrading acetylcholine.

The positive, albeit partial, success of Tacrine attests to thepotential value of improved anticholinesterase treatment of Alzheimer'sDisease. However, anti-acetylcholinesterase therapies for Alzheimer'sDisease require high doses of drug and produce side-effects associatedwith systemic cholinergic toxicity. Tacrine, for example, has beenassociated with liver damage and blood disorders in some patients[Johansson and Nordberg, 1993]. Further, current ACHE inhibitorsinteract non-specifically with the AChE-homologous serum proteinbutyrylcholinesterase and stimulate regulatory feedback pathways leadingto enhanced expression of AchE.

Although newer inhibitors such as E-2020 having greater specificity foracetylcholinesterase provide for lower doses [Rogers et al., 1996], theyare not likely to completely overcome the problem of cross-reactivitywith butyrylcholinesterase, given the high degree of similarity betweenthe two proteins [Loewenstein-Lichtenstein et al., 1996]. Moreover,liver function, red blood cell counts, and natural variations in thegenes encoding both acetylcholinesterase and butyrylcholinesterase willdetermine both the quantity and quality of the drug scavenging potentialamong individual patients. Several mutations in thebutyrylcholinesterase gene already have been suggested to create agenetic predisposition for adverse responses to anti-cholinesterases[Loewenstein-Lichtenstein et al., 1995]. This implies that even in thebest case scenario for acetylcholinesterase inhibitor-based therapies,various elements must be considered in designing individualized dosageregimens on a patient-by-patient basis.

Myasthenia gravis (MG) is an autoimmune disease in which antibodiesdirected against the nicotinic acetylcholine receptor (AChR) at themotor end plate of the neuromuscular junction (NMJ) by binding to theACHR impair neuromuscular communication either directly or through NMRdegradation. Therapeutic strategies for treating MG now includeanticholinesterase drugs, immunosuppressive drugs, thymectomy andplasmapheresis [see Myasthenia Gravis And Related Disorders, Annals ofthe New York Academy of Sciences, volumes 681 (1993), 505 (1987), 377(1981), 274 (1976) and 135 (1966) for an overview of the progress anddevelopment of the understanding of MG disease etiology and therapeuticstrategies as well as the 1998 volume (in press)].

AChE is accumulated at neuromuscular junctions (Salpeter 1967) where itserves a vital function in modulating cholinergic neurotransmission(reviewed by Soreq and Zakut, 1993). The molecular mechanisms by whichAChE and other synaptic proteins are targeted to the NMJ are poorlyunderstood. Compartmentalized transcription and translation in andaround the junctional nuclei probably contribute to the NMJ localizationof AChE (Jasmin et al., 1993). Therefore, anticholinesterase therapy isutilized with almost all patients in order to reduce AChE and therebyincrease the halflife of acetylcholine thereby potentiatingneuromuscular transmission. This therapy is often used in concert withimmunosuppressive therapy (generally steroids). It is the general goalto remove the patient from immunosuppressive therapy due to its sideeffects.

Pyridostigmine (Mestinon®) remains the drug of choice in treatingmyasthenics due to its effectiveness and tolerance by patients.Ambenonium (Mytelase®) is used for those MG patients who cannot toleratepyridostigmine. However, patients must be involved in the determinationof dosage of the drug since dosage will often need to be adjusted overany twenty-four hour period. Variables such as menstrual cycle,infections and emotional stress affect dosage and patients must betrained to modify their dosage to take these and other factors intoconsideration. Overdosage of pyridostigmine can lead to cholinergiccrisis and even with good patient education such overdoses occur.Additionally, as discussed herein above genetic predispositions topredisposition for adverse responses to anti-cholinesterases[Loewenstein-Lichtenstein et al., 1995] must be considered.

Cholinergic crisis due to anticholinesterase drug overdose ischaracterized by increasing muscle weakness which if involving therespiratory muscles can lead to a myasthenic crisis and death.Myasthenic crisis due to increase in severity of disease can alsopresent with the same symptoms. Distinguishing between the two isextremely important since treatment for a cholinergic crisis istermination of anticholinesterase drugs while the non-drug associatedmyasthenic crisis would indicate an increase in anti-cholinergic drugdosage. Therefore finding alternatives to anticholinesterase drugtherapy in MG would be useful.

These considerations indicate the need to develop a new generation ofanti-acetylcholinesterase drugs displaying increased target specificity,improved efficacy and reduced side effects.

Breakthroughs in molecular biology and the human genome project haveopened previously unforeseen possibilities for targeted interventionwith mammalian gene expression. These include permanent approaches suchas transgenic overexpression or recombinant disruption of specific genesas well as novel approaches for transient suppression of gene function.Short synthetic antisense (AS) oligodeoxynucleotides (AS-ODN) designedto hybridize with specific sequences within a targeted mRNA belong tothe latter class.

Many excellent reviews have covered the main aspects of antisensetechnology and its enormous therapeutic potential. The literaturenaturally progressed from chemical [Crooke, 1995] into cellular [Wagner,1994] and therapeutic [Hanania, et al, 1995; Scanlon, et al, 1995]aspects of this rapidly developing technology. Within a relatively shorttime, ample information has accumulated about the in vitro use of AS-ODNin cultured primary cells and cell lines as well as for in vivoadministration of such ODNs for suppressing specific processes andchanging body functions in a transient manner. This wealth ofaccumulated experience now offers a novel way to analyze the antisenseapproach, namely, to compare its in vitro uses with its in vivo ones[Lev-Lehman et al, 1997]. Further, enough experience is now available invitro and in vivo in animal models as shown in the Examples of thepresent application to predict human efficacy.

AS intervention in the expression of specific genes can be achieved bythe use of synthetic AS-ODNs [for recent reports seeLefebvre-d'Hellencourt et al, 1995; Agrawal, 1996; Lev-Lehman et al,1997]. AS-ODNs are short sequences of DNA (averaging 15-25 mer) designedto complement a target mRNA of interest and form an RNA:ODN duplex. Thisduplex formation can prevent processing, splicing, transport ortranslation of the relevant mRNA. Moreover, certain AS-ODNs can elicitcellular RNase H activity when hybridized with their target mRNA,resulting in mRNA degradation [Calabretta et al, 1996]. In that case,RNase H will cleave the RNA component of the duplex and can potentiallyrelease the AS-ODN to further hybridize with additional molecules of thetarget RNA. An additional mode of action results from the interaction ofAS-ODNs with genomic DNA to form a triple helix which may betranscriptionally inactive. See FIG. 1 for a schematic representation ofthe modes of action of AS-ODN.

Phosphorothioate antisense oligonucleotides do not show significanttoxicity and exhibit sufficient pharmacodynamic half-lives in animals[Agarwal et al., 1991, 1996]. Antisense induced loss-of-functionphenotypes related with cellular development were shown for the glialfibrillary acidic protein (GFAP), implicated in astrocyte growth withinastrocyte-neuron cocultures [Winstein et al., 1991], for themyelin-associated glycoprotein in Schwann cells, responsible forformation of the compact myelin sheath formation surrounding these cell[Owens and Bunge, 1991], for the microtubule-associated tau proteinsimplicated with the polarity of hippocampal neurons and their axonformation [Caceres and Kosik, 1990], for the β₁ -integrin, important forneuronal migration along radial glial cells, and for the establishmentof tectal plate formation in chick [Galileo et al., 1991] and for theN-myc protein, responsible for the maintenance of cellular heterogeneityin neuroectodermal cultures (ephithelial vs. neuroblastic cells, whichdiffer in their colony forming abilities, tumorigenicity and adherence)[Rosolen et al., 1990; Whitesell et al, 1991]. Antisense oligonucleotideinhibition of basic fibroblast growth factor (bFgF), having mitogenicand angiogenic properties, suppressed 80% of growth in glioma cells[Morrison, 1991] in a saturable and specific manner. The antisenseoligonucleotides were targeted against the initiation and splice sitesin bFgFmRNA, they reduced activity of the resulting protein and senseoligomers remained inactive. In soft-agar cultures, antisenseoligonucleotides reduced the size of glial colonies and inducedappearance of larger cells within them [Morrison, 1992]. Beinghydrophobic, antisense oligonucleotides interact well with phospholipidmembranes [Akhter et al., 1991]. Following their interaction with thecellular plasma membrane, they are actively transported into livingcells [Loke et al., 1989], in a saturable mechanism predicted to involvespecific receptors [Yakubov et al., 1989].

SUMMARY OF THE INVENTION

According to the present invention, synthetic nuclease resistantantisense oligodeoxynucleotides capable of selectively modulating humanacetylcholinesterase (AChE) production having the following sequencesare disclosed:

5'ACGCTTTCTTGAGGC 3' SEQ ID No:1,

5'GGCACCCTGGGCAGC 3' SEQ ID No:2,

5'CCACGTCCTCCTGCACCGTC 3' SEQ ID No:6, and

5'ATGAACTCGATCTCGTAGCC 3' SEQ ID No:7.

Further, additional synthetic nuclease resistant antisenseoligodeoxynucleotide are disclosed including

5'GCCAGAGGAGGAGGAGAAGG 3' SEQ ID No:4,

5'TAGCGTCTACCACCCCTGAC 3' SEQ ID No:5,

5'TCTGTGTTATAGCCCAGCCC 3' SEQ ID No:17, and

5'GGCCTGTAACAGTTTATTT 3' SEQ ID No:18.

A nuclease resistant antisense targeted against the splice junction inthe AChEmRNA post splice message is disclosed. In an embodiment theE4-E6 junction in the E2-E3-E4-E6 splice variant AChEmRNA is disclosedas being highly specific for the muscle and central nervous systemsplice variant of AChE.

Synthetic nuclease resistant artisense oligodeoxynucleotides capable ofselectively modulating human acetylcholinesterase production in thecentral nervous system or capable of selectively reducing humanacetylcholinesterase deposition in the neuromuscular junction having thefollowing sequences are also disclosed:

5'ACGCTTTCTTGAGGC 3' SEQ ID No:1,

5'GGCACCCTGGGCAGC 3' SEQ ID No:2

5'CCACGTCCTCCTGCACCGTC 3' SEQ ID No:6,

5'ATGAACTCGATCTCGTAGCC 3' SEQ ID No:7,

5'GCCAGAGGAGGAGGAGAAGG 3' SEQ ID No:4,

5'TAGCGTCTACCACCCCTGAC 3' SEQ ID No:5,

5'TCTGTGTTATAGCCCAGCCC 3' SEQ ID No:17, and

5'GGCCTGTAACAGTTTATTT 3' SEQ ID No:18.

The present invention also discloses pharmaceutical or medicalcomposition comprising as active ingredient at least one of thesesynthetic nuclease resistant antisense oligodeoxynucleotides in aphysiologically acceptable carrier or diluent.

The present invention further discloses a method to restore balancedcholinergic signaling in the brain in patients in need of suchtreatment, particularly relating to learning and memory as well asstress disorders and Parkinson's. The method includes the steps ofadministering to a patient in need of such treatment a therapeuticallyeffective amount of at least one of the synthetic nuclease resistantAS-ODN of the present invention.

The present invention further discloses a method to reduce productionand therefore deposition of acetylcholinesterase in the neuromuscularjunction in patients in need of such treatment, particularly patientswith myasthenia gravis. The method includes the steps of administeringto a patient in need of such treatment a therapeutically effectiveamount of at least one of the synthetic nuclease resistant antisenseoligodeoxynucleotides of the present invention.

This technology specifically arrests the production, as opposed tobiochemical activity, of acetylcholinesterase in brain cells andprevents and/or reduces deposition at the neuromuscular junction. Thistechnology is based on disruption of the pathway leading toacetylcholinesterase biosynthesis by administration of very low doses ofantisense oligonucleotides. Antisense oligonucleotides are uniquelytargeted against the gene encoding acetylcholinesterase rather than theultimate gene product (i.e. the protein). Therefore, the moleculartarget of these antisense oligonucleotides against acetylcholinesteraseneither interact with the related enzyme butyrylcholinesterase norsuppress butyrylcholinesterase gene expression. Hence, this potentialdrug works effectively at low doses while avoiding many of the sideeffects associated with Tacrine and related cholinergic drugs forAlzheimer's disease and pyridostigmine and related drugs for Myastheniagravis.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a schematic diagram of the modes of action of antisense (AS)oligodeoxynucleotides (ODN) showing a gene being transcribed into MRNAand following uptake of AS-ODN both inhibition of transcription throughtriple helix formation, interference with RNA splicing or translationmay occur or the RNA:ODN duplex can elicit RNase H activity resulting inRNA degradation and preventing protein production.

FIG. 2 is a chart with photographs of gels inserted showing reduction inAChE mRNA levels in the cortex of mice treated with antisenseoligodeoxynucleotides. Specific primers were employed to detect hACHE,mACHE or synaptophysin (Syn) mRNAs; cDNA product was collected everythird cycle between cycles 21-36, subjected to gel electrophoresis andstained with ethidium bromide. The products from cycles 21-36 arepresented in the figure from left to right. Levels of AChE activity incortex of mice injected with buffer or with AS oligodeoxynucleotides arepresented in nmol substrate hydrolyzed/min/ug protein

FIG. 3 is a graph showing that antisense oligonucleotides injectedi.c.v. gives a reduction in AChE catalytic activity in subcorticalregions. Each circle represents the AChE activity measured in thesubcortical region of a single injected mouse. The column ofbuffer-injected mice represents data from two independent experimentsperformed on age-matched mice. The average activity calculated for eachgroup is indicated by a horizontal line.

FIGS. 4A-B demonstrates the in vivo antisense suppression of ACHEmRNAwherein FIG. 4A schematically presents the mouse ACHE gene with itspromoter (P), 6 exons (numbered 1-6) and 4 introns. Alternative splicingyields 3 variant mRNA transcripts that encode polypeptides differing intheir C-terminal peptide sequences. FIG. 4B is a photograph of the gelelectrophoresis of PCR products at various cycles and demonstrates theeffect on total ACHEmRNA of antisense (AS) oligonucleotides targetedagainst the common exon E2 (mE2) or the alternative exon E5 (mE5)compared with those of sense (S) oligos based on the homologous humanACHE gene sequence or sham injections with PBS. β-actin mRNA served as acontrol for non-specific effects on transcription. Note that both AS-mE2and AS-mE5 exert specific reduction of E6-containing ACHEmRNA in bonemarrow but not muscle at the administered doses while actin mRNA wasunaffected by any treatment. Gels present data from a singlerepresentative animal among three treated individuals.

FIGS. 5A-B shows anti-ACHE ODNs and their targeted ACHEmRNA sequenceswherein FIG. 5A is a photograph of a gel of RT-PCR amplificationproducts derived from total RNA preparations of adult (2 months) mousebrains, cerebral primary neurons from mouse embryos (embryonic day 13)grown in culture for 3 days with or without 0.5 μg/ml actinomycin D(Act. D) or non-differentiated PC12 cells. Shown are 10% of theproducts, resolved by electrophoresis on agarose slab gels and stainedwith ethidium bromide, of RT-PCR amplification of 200 ng RNA incubatedwith selective primers for the ACHEmRNA transcripts 3'-terminated withE5, E6 or I4/E5 sequences [for details, see Karpel et al., 1996]. Notethat the E6-ACHEmRNA transcript is the most pronounced of all in each ofthese sources, and that it remains largely intact following 3 days inthe presence of actinomycin D in the absence of novel transcripts. PC12cells, like murine brain neurons, express 3 alternative ACHEmRNAs. FIG.5B is a schematic diagram of the location and various parameters of theAS-ODNs. Location of each of the AS-ODNs ODNs (1-7) (bold underlines) ismarked along the ACHE gene, which is represented schematically. Emptyboxes depict introns (I) and filled boxes, exons (E) with the exceptionof pseudointron 4 (I4) which is also shaded. The broken lines underneathdenote alternative splicing options. Open reading frame (ORF) regionsare marked by a solid line above, initiated by the first AUG codon atthe 5'-end of the gene. ODN structures are classified into those with nopredicted secondary structure (N) and those predicted to form loops(drawn). G, C contents are also noted. Predicted melting temperaturesand free energies of the ODNs are shown below each of their positions(PRIMER program, University of Wisconsin GCG software package.)

FIGS. 6A-B shows the neurotoxicity of the AS-ODNs wherein FIG. 6A is abar graph of the survival rate of undifferentiated PC12 cells after 24hours in the presence of either 1 μM (filled bars) or 10 μM (open bars)of each of the ODNs. Standard error of the mean for 3 cultures is shownby the error bars. Note the relatively higher toxicity of AS2, even at 1μM, and the increased neurotoxicity at 10 μM of most other ODNs. FIG. 6Bis a graph showing the linear relationship between cell number and freethiol croups. The number of non-differentiated PC12 cells deposited inmicrotiter wells was measured by phase microscopy and manual counting ina haemocytometer. Shown is the average absorption at 405 nm per 1,000cells for six cultures that were exposed to buffered Triton X-100 andDTNB.

FIG. 7 is a graph showing the efficacy of AS-ODNs at 1 μM depends on NGFinduction but not on their target position along the coding region inthe ACHEmRNA sequence. Shown are percent inhibition of AChE activity inuntreated cultures. Values of AChE in NGF-treated cultures are in filledcircles and those for non-differentiated PC12 cells are in emptycircles. The data points for each AS-ODN are located below theirpositions in the ACHEcDNA sequence presented schematically above thegraph. Error bars show the standard errors of the mean for 3 wells ineach test. The values corresponding to AS5 are located in a separate boxto the right, under the alternative E5 exon. Note that for most of theAS-ODNs, inhibition efficacies are higher in the NGF-treated than innon-treated cultures.

FIGS. 8A-B are graphs of the semi-quantitative measurement of AChE mRNAby kinetic follow-up of RT-PCR. RT-PCR analyses were performed for mRNAsfor AChE (A) and actin (B). Amplification products of total RNAextracted from untreated differentiated PC12 cells (none) or cellstreated with ODNs (AS1, AS3, AS4, AS6 or AS-B) were subjected to gelelectrophoresis and CCD quantification. Shown are percent of maximalfluorescence intensities of 12 μl of ethidium bromide-stained productscollected at cycles 18, 20, 22, 24, 26, 28 (for actin mRNA) and 26, 28,30, 32, 34, 36 (for AChE mRNA). Inset: linear regression analyses ofaccumulation kinetics were performed only for those time points whenproduct accumulation proceeded at constant pace (cycles 28, 30 and 32for AChE mRNA, cycles 20, 22, 24 for actin mRNA). Note the shift to theright in the curves derived for AS-ODN treated cells as compared withcontrol cells, and the absence of such shift in the actin mRNA curves.

FIGS. 9A-B are bar graphs of deficient performance of AChE transgenicmice in social exploration test (A) corrected by Tacrine (B). FIG. 9Ashows adult transgenic or control mice exposed to an unknown juvenileand the time invested in olfactory recognition recorded (t1). Followingthe indicated intervals (in minutes) each mouse was presented with thesame, or a different, juvenile, and the recognition time noted (t2).Presented are the average±SD for t2/t1 for groups of 5-8 mice. Asteriskindicates statistically significant differences in t2 vs. t1. Note thattransgenic mice lost the ability to recognize the "same" mouse within 10minutes compared with 30 minutes for controls. FIG. B represents theimproved memory performance observed among transgenic mice following asingle administration of tacrine (1 mg/g wt) and a 20 minute intervalbetween exposures.

FIG. 10 is a bar graph of deficient performance of AChE transgenic micein a taste aversion test demonstrating that hAChE transgenic mice learnbut do not have long-term memory.

FIG. 11 is a is a schematic diagram of the three splice variants ofAChE.

FIG. 12 shows the amino acid sequences of human (H) AChE variants fromthe end of E4 to the end of the protein in the three variants, E1-4,6(SEQ ID No:21), E1-5 (SEQ ID No:22), E1-4-I4-E5 (readthrough; SEQ IDNo:23).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a synthetic nuclease resistant antisenseoligodeoxynucleotide (AS-ODN) capable of selectively modulating humanacetylcholinesterase (AChE) production in the central nervous system(CNS) and neuromuscular junction (NMJ). The term modulating as usedherein refers to selective inhibition and/or stimulation ofacetylcholinesterase production, that is an interaction capable ofchanging the rate of, or stopping, production.

Such selective modulation, i.e. changes in rate of production, can leadfor example to (1) changes in neuronal activity, (2) changes in learningand memory, or (3) reduction of AChE in the NMJ, i.e, a reduction indeposition of AChE in the NMJ.

The specific sequence of the AS-ACHE-ODN is determined and tested forefficacy as described herein below. The sequence is selected such thatit is targeted to a splice variant of the AChEmRNA that isactive/predominant in the central nervous system and/or muscle therebyreducing or eliminating the AS-ODN activity in other tissues. The targetsequence is selected so as to be accessible to the AS-ODN and unique tothe splice variant in the target tissue. It is possible to select asequence that while not unique to the splice variant is not accessibleto the AS-ODN in other tissues (see Examples). In summary, the AS-ODNsare non-toxic, highly selective for the ACHE gene, operate in asequence-dependent manner and can be targeted to specific tissue and/orcells.

In an embodiment the antisense oligodeoxynucleotide has one of thefollowing sequences:

5'ACGCTTTCTTGAGGC 3' SEQ ID No:1,

5'GGCACCCTGGGCAGC 3' SEQ ID No:2,

5'GCCAGAGGAGGAGGAGAAGG 3' (hAS-1) SEQ ID No:4,

5'TAGCGTCTACCACCCCTGAC 3' (hAS-2) SEQ ID No:5,

5'CCACGTCCTCCTGCACCGTC 3' (hAS-3) SEQ ID No:6,

5'ATGAACTCGATCTCGTAGCC 3' (hAS-4) SEQ ID No:7,

5'TCTGTGTTATAGCCCAGCCC 3' (hAS-6) SEQ ID No:17,

5'GGCCTGTAACAGTTTATTT 3' (hAS-7) SEQ ID No:18.

SEQ ID No:1 is directed against the human ACHE sequence starting atposition 1119 (for numbering of nucleotides see Soreq et al, 1990). SEQID No:2 is directed against the human ACHE sequence starting at position1507. SEQ ID Nos:4-7 and 17-18 are the human equivalent for the mASsequences tested in Examples 4-8 and correspond to mAS-1-4,6-7 (SEQ IDNos: 8, 10, 11, 12, 13, 14 respectively). Note that there is littlehomology between the mAS3 and hAS3 antisense sequence due to the lack ofhomology between the two species at that position in the sequence.Control hAS sequences were inverse hAS-1 5'GGAAGAGGAGGAGGAGACCG3' (SEQID No;19) and inverse hAS-6 5'CCCGACCCGATATTGTGTCT3' (SEQ ID No:20).

The term "oligonucleotide" refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted oligomers comprising non-naturally occurringmonomers or portions thereof, which function similarly. Incorporation ofsubstituted oligomers is based on factors including enhanced cellularuptake, or increased nuclease resistance and are chosen as is known inthe art. The entire oligonucleotide or only portions thereof may containthe substituted oligomers.

Antisense intervention in the expression of specific genes can beachieved by the use of synthetic antisense oligonucleotide sequences[for recent reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal,1996; Lev-Lehman et al, 1997]. Antisense oligonucleotide sequences maybe short sequences of DNA, typically 15-30 mer but may be as small as 7mer [Wagner et al, 1996], designed to complement a target mRNA ofinterest and form an RNA:AS duplex. This duplex formation can preventprocessing, splicing, transport or translation of the relevant mRNA.Moreover, certain AS nucleotide sequences can elicit cellular RNase Hactivity when hybridized with their target mRNA, resulting in mRNAdegradation [Calabretta et al, 1996]. In that case, RNase H will cleavethe RNA component of the duplex and can potentially release the AS tofurther hybridize with additional molecules of the target RNA. Anadditional mode of action results from the interaction of AS withgenomic DNA to form a triple helix which may be transcriptionallyinactive.

Antisense induced loss-of-function phenotypes related with cellulardevelopment were shown for the glial fibrillary acidic protein (GFAP),for the establishment of tectal plate formation in chick [Galileo etal., 1991] and for the N-myc protein, responsible for the maintenance ofcellular heterogeneity in neuroectodermal cultures (ephithelial vs.neuroblastic cells, which differ in their colony forming abilities,tumorigenicity and adherence) [Rosolen et al., 1990; Whitesell et al,1991]. Antisense oligonucleotide inhibition of basic fibroblast growthfactor (bFgF), having mitogenic and angiogenic properties, suppressed80% of growth in glioma cells [Morrison, 1991] in a saturable andspecific manner.

Nuclease resistance, where needed, is provided by any method known inthe art that does not substantially interfere with biological activityof the antisense oligodeoxynucleotides as needed for the method of useand delivery [Iyer et al., 1990; Radhakrishnan, et al., 1990; Eckstein,1985; Spitzer and Eckstein, 1988; Woolf et al., 1990; Shaw et al.,1991]. Modifications that can be made to antisense oligonucleotides inorder to enhance nuclease resistance include modifying the phosphorousor oxygen heteroatom in the phosphate backbone, short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. These include preparing2'-fluoridated, O-methylated, methyl phosphonates, phosphorothioates,phosphorodithioates and morpholino oligomers. In a non-limitingembodiment it is provided by having phosphorothioate bonds linkingbetween the four to six 3'-terminus nucleotide bases. Alternatively,phosphorothioate bonds link all the nucleotide bases. Phosphorothioateantisense oligonucleotides do not normally show significant toxicity atconcentrations that are effective and exhibit sufficient pharmacodynamichalf-lives in animals [Agarwal et al., 1996] and are nuclease resistant.Alternatively the nuclease resistance can be provided by having a 9nucleotide loop forming sequence at the 3'-terminus having thenucleotide sequence CGCGAAGCG (SEQ ID No:3). The use of avidin-biotinconjugation reaction can also be used for improved protection of AS-ODNsagainst serum nuclease degradation [Boado and Pardridge, 1992].According to this concept the AS-ODN agents are monobiotinylated attheir 3'-end. When reacted with avidin, they form tight,nuclease-resistant complexes with 6-fold improved stability overnon-conjugated ODNs.

Studies of others have shown extension in vivo ofAS-oligodeoxynucleotides [Agarwal et al., 1991]. This process,presumably useful as a scavenging mechanism to remove alienAS-oligonucleotides from the circulation depends on the existence offree 3'-termini in the attached oligonucleotides on which the extensionoccurs. Therefore partial phosphorothioate, loop protection orbiotin-avidin at this important position should be sufficient to ensurestability of these AS-oligodeoxynucleotides.

The present invention also includes all analogues of, or modificationsto, an oligonucleotide of the invention that does not substantiallyaffect the function of the oligonucleotide. Such substitutions may beselected, for example, in order to increase cellular uptake or forincreased nuclease resistance as is known in the art. The term may alsorefer to oligonucleotides which contain two or more distinct regionswhere analogues have been substituted.

The nucleotides can be selected from naturally occurring orsynthetically modified bases. Naturally occurring bases include adenine,guanine, cytosine, thymine and uracil. Modified bases of theoligonucleotides include xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halocytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil,8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines,8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxylguanine and other substituted guanines, other aza and deaza adenines,other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluorocytosine.

In addition, analogues of nucleotides can be prepared wherein thestructure of the nucleotide is fundamentally altered and that are bettersuited as therapeutic or experimental reagents. An example of anucleotide analogue is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogues have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. Further, PNAs have beenshown to bind stronger to a complementary DNA sequence than a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, morpholinopolymer backbones [U.S. Pat. No. 5,034,506], cyclic backbones, oracyclic backbones, sugar mimetics or any other modification includingwhich can improve the pharmacodynamics properties of theoligonucleotide.

The synthetic nuclease resistant antisense oligodeoxynucleotides of thepresent invention can be synthesized by any method known in the art. Forexample, an Applied Biosystems 380B DNA synthesizer can be used. Finalpurity of the oligonucleotides is determined as is known in the art.

The present invention also discloses a pharmaceutical or medicalcomposition comprising as active ingredient at least one syntheticnuclease resistant antisense oligodeoxynucleotides capable ofselectively modulating human acetylcholinesterase production in thecentral nervous system or neuromuscular junction in a physiologicallyacceptable carrier or diluent. In one preferred embodiment the syntheticnuclease resistant antisense oligodeoxynucleotide is SEQ ID Nos:1, 2 and7, however SEQ ID Nos:4-6, 17 and 18 can also be used in the appropriatetarget.

The present invention also provides a method to restore balancedcholinergic signaling in the central nervous system in patients in needof such treatment such as those with deficits in memory and learing orin stress disorders and Parkinson's. The method comprises administeringto a patient in need of such treatment a therapeutically effectiveamount of at least one of a synthetic nuclease resistant AS-ODN capableof selectively modulating human acetylcholinesterase production in thecentral nervous system in a physiologically acceptable carrier ordiluent. The present invention also provides a method to reducedeposition of acetylcholinesterase in the neuromuscular junction inpatients in need of such treatment comprising administering to a patientin need of such treatment a therapeutically effective amount of at leastone of a synthetic nuclease resistant AS-ODN capable of selectivelymodulating human acetylcholinesterase production in the neuromuscularjunction in a physiologically acceptable carrier or diluent.

Acceptable carriers, exipients are nontoxic to recipients at the dosagesand concentrations employed, and include buffers, such asphysiologically acceptable buffers such as phosphate buffered saline,and more generally all suitable carriers known in the art. Thecompositions may further optionally contain physiologically acceptableadditives such as antioxidants; mono- and disaccharides; salt-formingcounterions such as sodium and/or nonionic surfactants. Sustainedrelease compositions are also contemplated within the scope of thisapplication. These may include semi-permeable polymeric matrices in theform of shaped articles such as films or microcapsules. The antisenseoligodeoxynucleotides and compositions of the invention must be sterile.Antisense delivery has also been shown using liposomes [Juliano andAkhtar, 1992].

In one embodiment the synthetic nuclease resistant antisenseoligodeoxynucleotide is SEQ ID Nos:1, 2, 6 and 7, however SEQ IDNos:4-5, 17 and 18 can also be used in the appropriate target.

An important feature of the present nuclease resistant antisenseoligodeoxynucleotide invention is that they can be administered bysimple subcutaneous, intramuscular, intravenous or intraperitonealinjection and that their effects last for at least several weeks. Thelimited toxicity of the AS-ODNs of the present invention is ofparticular importance for their therapeutical uses.

The AS-ODN is administered and dosed in accordance with good medicalpractice, taking into account the clinical condition of the individualpatient, the site and method of administration, scheduling ofadministration, patient age, sex, body weight and other factors known tomedical practitioners. The pharmaceutically "effective amount" forpurposes herein is thus determined by such considerations as are knownin the art. The amount must be effective to achieve improvementincluding but not limited to changes in levels of AChE in the CNS orneuromuscular junction, or improvement or elimination of symptoms andother indicators as are selected as appropriate measures by thoseskilled in the art.

For specific delivery within the CNS intrathecal delivery can be usedwith, for example, an Ommaya reservoir. U.S. Pat. No. 5,455,044 providesfor use of a dispersion system for CNS delivery or see U.S. Pat. No.5,558,852 for a discussion of CNS delivery. In addition, pharmacologicalformulations that cross the blood-brain barrier can be administered.[Betz et al., 1994; Brem et al., 1993]. Such formulations can takeadvantage of methods now available to produce chimeric molecules inwhich the present invention is coupled to a brain transport vectorallowing transportation across the barrier [Pardridge, et al., 1992;Pardridge, 1992; Bickel, et al., 1993]. Further, Applicant's in aco-pending application U.S. patent application Ser. No. 08/975,084 filedNov. 20, 1997 assigned to the same assignee and incorporated in itsentirety herein by reference have demonstrated that a stress-mimickinginducing agent or treatment will open the barrier such as the ACHE-I4readthrough splice variant or the I4 peptide or adrenaline or atropine.

For modulating the production and therefore deposition of AChE at theneuromuscular junction, AS-ODNs which target the muscle form are used.In general, being hydrophobic antisense oligonucleotides interact wellwith phospholipid membranes [Akhter et al., 1991]. Following theirinteraction with the cellular plasma membrane, they are activelytransported into living cells [Loke et al., 1989], in a saturablemechanism predicted to involve specific receptors [Yakubov et al.,1989]. Therefore, AS-ODN are available to the muscle cells which produceAChE that is deposited (transported, released to) the neuromuscularjunction.

As with any drug, testing the potential therapeutic utility of antisenseoligonucleotides targeted against acetylcholinesterase requires anappropriate model, either in vivo, ex vivo or in vitro. Since mice donot naturally develop a disease resembling human dementia, Applicantshave generated a unique transgenic mouse model for Alzheimer's Diseaseto serve this purpose [Beeri et al., 1995 and co-pending U.S. patentapplication Ser. No. 08/370,156 assigned to the same assignee andincorporated in its entirety herein by reference]. These geneticallyengineered mice overproduce human acetylcholinesterase in cholinergicbrain cells. Excess acetylcholinesterase in brain cells induceacetylcholine shortages similar to those assumed to promote thecognitive dysfunction associated with Alzheimer's Disease. And, indeed,Applicants transgenic mice display age-dependent deterioration incognitive performance as initially measured by a standardized swimmingtest for spatial learning and memory, a social recognition test as setforth in Example 7 herein below and a taste aversion test. Since theexcess acetylcholinesterase in the brains of these mice is derived fromhuman DNA, it is susceptible to antisense oligonucleotides targetedagainst the human acetylcholinesterase gene. This animal system andbrain slices derived thereof, therefore provides the ability to testanti-acetylcholinesterase antisense technology by in vivo, ex vivo andin vitro means to restore balanced cholinergic signaling in the brainand thereby relieve some of the impaired cognitive function from whichAlzheimer's Disease patients suffer and to test the efficacy oftreatment initiated at pre-symptomatic stages. In general, initialscreening for efficacy occurs ex vivo or in vitro, preferably in brainslices. Following this screening the AS-ODN is tested in the hACHEtransgenic mice for efficacy. Suitable candidates for human testing arethereby determined. This model system also responds to Tacrine in thesame manner as humans (see Examples) thereby also supporting its use asa model system for testing AS-ODNs.

For testing of AS-ODN in myasthenic animals, the Experimental AutoimmuneMyasthenia Gravis model is available. Further, Applicants have shown[Andres et al, 1997] via in vivo behavioral tests (rope-gripping) and insitu histology (synapses have an MG-like structure) a mimicking modelfor MG in transgenic mice that overexpress AChE which can be used intesting new AS-ODNs.

Applicants have established protocols for in vivo administration ofoligonucleotides using intravenous, intraperitoneal, and directintracerebroventricular (i.c.v.) routes. Results show the efficacy ofnuclease resistant antisense oligonucleotides in reducing AChE catalyticactivity in brain tissue of transgenic mice. These studies provide thebasis for testing and defining therapeutically useful forms and doses ofoligonucleotides in vivo.

As shown in the Examples, AS-ACHE-ODNs have been produced and injectedwhich are targeted against both human and mouse AChEmRNA (see Tables Iand II). AS-ODNs were nuclease resistant as indicated.

No acute toxic effects were observed in any AS-ODN treated humantransgenic mouse and behavior appeared normal in all treated animals.AS-ODN targeted against hAChEmRNA resulted in diminished levels of bothhAChE- and mAChE mRNAs (FIG. 2) and dramatically reduced protein levelsin one of two animals. AS-ODN against mAChEmRNA resulted in a 3 cycledelay in appearance of RT-PCR product in one animal (approx 8-foldreduction in mRNA). When 100 pmole (approx 1 ug) AS-ODN against hAChE-or mAChE- mRNA was delivered i.c.v. to 15 day old mice, 2 of 3 mice ineach group displayed total AChE activities >1 S.D. below the meanactivity measured in buffer injected animals 40 hours post injection(FIG. 3).

In designing AS-ODNs for ACHEmRNA in target cells it is necessary todefine which of the three alternative transcripts expressed in mammalsis present in these cells. PCR amplification using primers selective foreach of the transcripts determines which are present and in whatintensity. In general the transcript with the highest intensity in thetarget tissue is selected. As shown in the Examples there are tissuedifferences (see FIGS. 4 and 5). As previously discussed the dominantcentral nervous system and muscle AChE (AChE-T) found in theneuromuscular junction (NMJ) is encoded by an mRNA carrying exon E1 andthe invariant coding exons E2, E3, and E4 spliced to alternative exon E6[Li et al., 1991; Ben Aziz-Aloya et al., 1993]. As shown in theExamples, it is possible that an AS targeted to a specific Exon willtarget both pre- and post-splice AChEmRNA. In order to specificallytarget the post-splice AChEmRNA the unique splice junction of the threeforms can be targeted by the antisense (FIGS. 11-12) after splicing hasoccurred. A 20 mer AS-ODN is preferred but other lengths are used thatare complementary to the unique sequence at the splice junction. Theantisense seqeunces generally extend 10 nucleotides on either side ofthe junction, but other ranges on each side can be used as long as theydefine the unique splice junction sequence. In an embodiment an AS-ODNtargeted against the E4-E6 junction sequence (FIG. 12) will be generallyspecific for the central nervous system and neuromuscular junction form(AChE-T) as shown in the Examples.

Interestingly, as described herein the mAS-ODNs, except mAS6 and mAS7,were targeted against translationable sequences included in the openreading frame of ACHEmRNA. mAS7, targeted to the 3'-region of exon 6,was significantly less effective than those designed against thesequence common to all alternatively-spliced ACHEmRNA transcripts. Thisis not a general rule; on the contrary, AS-ODNs against 3'- regions inother mRNAs were shown to effectively induce destruction of the entiremRNA sequence [e.g. Bennet et al., 1994]. However, mammalian ACHEmRNA isespecially rich in G,C base pairs (67% in human ACHE, Soreq et al.,1990). Therefore, it is likely to be tightly folded. Since a truncatedhuman ACHEmRNA bearing only exons 2, 3 and 4 was found to betranslatable in Xenopus embryos [Seidman et al., 1997], it is possiblethat E6-ACHEmRNA is so tightly folded that RNaseH action on its 3'-exondoes not lead to destruction of exons 2, 3 and 4, leaving an mRNA whichencodes a catalytically active, C-terminally truncated protein.Therefore, an AS directed against the E4-E6 junction sequence will beeffected where one directed against the 3' region may not be.

The above discussion provides a factual basis for the use of AS-ODN. Themethods used with and the utility of the present invention can be shownby the following non-limiting examples and accompanying figures.

EXAMPLES

General Methods

General Methods in Molecular Biology

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,New York (1989, 1992), and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).Polymerase chain reaction (PCR) was carried out generally as in PCRProtocols: A Guide To Methods And Applications, Academic Press, SanDiego, Calif. (1990).

Reactions and manipulations involving other nucleic acid techniques,unless stated otherwise, were performed as generally described inSambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, and methodology as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference.

Synthesis of Antisense Oligodeoxynucleotides

Oligodeoxynucleotides were synthesized on an Applied Biosystems 380B DNAsynthesizer using phosphoramidites from the same company according tothe manufacturer's instructions. They were purified by reverse phaseHPLC on a Waters dual pump 6000A system in combination with a Watersautomated gradient controller and a model 481 UV spectrophotometeroperated at 260 nm with the 5'-protecting dimethoxytrityl group stillattached to the oligodeoxynucleotides. This was removed by standardtreatment with 80% aqueous acetic acid. The oligodeoxynucleotidesobtained were checked for purity again by HPLC.

For nuclease resistance where phosphorothioate groups whereincorporated, the oxidation step employing iodine was replaced byreaction with 3H-1,2-benzodithiol-3-one 1, 1-dioxide [Iyer et al.,1990]. This treatment protects the oligodeoxynucleotides againstnuclease [Eckstein, 1985; Spitzer and Eckstein, 1988] and prolongs theirduration in vivo [Woolf et al., 1990; Shaw et al., 1991]. Whereverpartial protection was required, reaction with 3H-1,2-benzodithiol-3-one1,1-dioxide was performed for the first three steps only, after whichregular synthesis was continued. The resultant partially protectedoligodeoxynucleotides were therefore blocked by phosphorothioate groupsonly in the last three internucleotidic bonds at their 3'-terminus.

For inclusion of a loop, the synthesis of the oligodeoxynucleotide wasextended at the 3' end to include SEQ ID No:3.

The antisense oligodeoxynucleotides were kept in 4 mM concentration at-20° C. and were diluted in phosphate buffered saline (PBS) prior totheir administration to mice.

Example 1 Summary of Prior Work with AS-ACHE-ODN in the HematopoieticSystem

Because of its unique properties, the hematopoietic system isparticularly well suited for antisense intervention with multiplecellular and molecular processes. The rapid proliferation and short halflife of hematopoietic cells as well as the efficient uptake andaccessibility of AS-ODNs in them are among the reasons for theseefficient effects of AS-ODNs in hematopoietic cells [Calabretta et al.,1996; Gerwirz et al, 1993].

To examine the role of AChE in controlling hematopoietic composition ingeneral and megakaryocytic (MK) development in particular, mature femalemice were treated in vivo with phosphorothioate AS-ACHE. To monitor theeffects of this treatment, bone marrow differential cell counts werecombined with a kinetic follow-up of polymerase chain reaction products(RNA-PCR) in different tissues [Lev-Lehman et al., 1994]. In situhybridization with ³⁵ S-labeled ACHE and BCHE cRNA probes, followed bycomputerized quantification of the hybridization data was used toassociate mRNA levels with specific cell types. The RNA-PCR analysisdemonstrated an apparently total abolition of ACHE mRNA at 12 dayspost-treatment, when lymphocyte and erythroid fractions were drasticallyreduced in the bone marrow of treated mice. This implicates ACHE in thedevelopment of both lymphocytes and erythrocytes, two cell lineagesexpressing this enzyme as well as showing the effectiveness of AS-ODNtreatment.

Because of their small numbers and longevity, it would not beinformative to evaluate differences in the MK fraction at day 12 sincepart of the MK would still represent cells from the pre-treatmentperiod. However, the secondary decrease in actin mRNA in the bonemarrow, where MK are replete with this mRNA species was taken as anindication of decrease in MK as well. As MK and erythroid cells areconsidered to share a common progenitor, these findings further suggestthat these progenitors are similarly affected by the AS-ACHE treatment.

Lymph nodes were selected as an additional tissue for the RT-PCRexperiments since this tissue is subject to a continuous replacement,similarly to bone marrow cells. The drastic decrease in lymph node ACHEmRNA levels 12 days post-treatment demonstrated efficient tissuedistribution of the administered AS-ACHE oligo.

These findings demonstrate transient changes in hematopoietic cellcomposition following AS-ACHE treatment, and in particular--increasedmyeloid fraction [Patinkin, et al., 1990, 1994; Lev-Lehman et al.,1994].

The in vivo effects of AS-ACHE oligonucleotides in increasing themyeloid fraction in bone marrow as discussed herein above, could reflectexpansion of progenitors, which could first be evident by an increase inthe faster-developing myeloid cells. Additionally or alternatively, itcould be due to enhanced myeloidogenesis or suppressed erythropoiesis.To distinguish between these possibilities, and to more closelyinvestigate the function of the ACHE gene in hematopoiesis, AS-ACHE wasadministered ex-vivo to primary hematopoietic cells. Its effects on geneexpression, expansion of progenitors, and differential cell compositionon mouse CFU-MK and CFU-GEMM colonies was examined [Soreq et al., 1994].These experiments, as well, resulted in an increase in the fraction ofmyeloid cells, reflecting both expansion of progenitors and increase inthe development of their progeny cells [Soreq et al., 1994].

The ex vivo experiments, using primary murine bone marrow cultures,provide an additional advantage over the in vivo ones in that theeffects of growth factors can be studied individually. For example, insuch primary cultures interleukin 3 (IL-3) induces expansion of afraction of the existing pluripotent stem cells into multipotentprogenitors, which can differentiate into megakaryocyte colony-formingunits (CFU-MK) composed of granulocytes, megakaryocytes, and macrophages[Patinkin et al., 1990; Lapidot-Lifson et al., 1992]. Addition oferythropoietin and transferrin to IL-3 and longer incubation timesinduce CFU-GEMM colonies, which contain granulocytes, erythroid cells,megakaryocytes, and macrophages. This implies that colony counts reflectexpansion and survival of progenitors that have given rise to progeny,whereas cell numbers reflect proliferation rates, and differential cellcompositions demonstrate which cell lineages developed and which wereprogrammed to die. Interference with expression of hematopoieticallyimportant genes by AS-ODN agents [Stein and Cheng, 1993] can conceivablyalter any or all of the characteristics of these cultures, and asapplicants have shown AS-ODNs targeted to cdc kinases [Lapidot-Lifson etal., 1992] and to the ACHE-related gene BCHE [Lapidot-Lifson et al.,1989; Soreq and Zakut, 1993], impair megakaryocytopoiesis in CFU-MKcolonies [Lapidot-Lifson et al., 1992; Patinkin et al., 1994; Soreq etal., 1994].

Example 2 Summary of Prior Work with AS-BCHE-ODN in the HematopoieticSystem

The role of BuChE in hematopoiesis was studied by comparing the effectsof AS-BCHE ODN administered to primary murine bone marrow cultures tothose observed for AS-ACHE ODNs. The findings demonstrated certainenhancement in myeloid cell fractions and corresponding suppression ofthe megakaryocyte fractions in both CFU-MK and CFU-GEMM culturesadministered with AS-BCHE ODNs. This erythropoietin-independent effectwas sequence-dependent and not associated with general apoptoticchanges. Complementary in vivo studies revealed continuation of theantisense-inducted destruction of BCHEmRNA for over 2 weeks, no effecton megakaryocytes survival and ex-vivo suppression of CFU-MK expansioncapacity following the in vivo treatment. Thus, AS-ACHE and AS-BCHEagents can be expected to exert similar effects on megakaryocytopoiesisalthough they do not cross-react with each other's target.

To avoid non-specific cytotoxicity of the oligonucleotides, partialphosphorothioated was used to protect the relevant oligos, replacingonly the three 3'-terminal internucleotidic bonds with phosphorothioategroups [Ehrlich et al., 1994]. Demonstration of a non-disturbedapoptotic index in experimental cell cultures, evidenced in unchangedladders of fragmented DNA, indicated that the studied effects did notresult from non-specific induction of programmed cell death. This, inturn, suggests that the increase in myeloid cell fraction was primarilydue to selective destruction of the target BCHEmRNA and the AS-ODNs.

Other experiments in this series demonstrated non-sequence dependenteffects of AS-ODE agents over hematopoiesis ex vivo. In both CFU-MK andCFU-GEMM cultures, partially protected AS-BCFE but not the senseoriented sequence S-BCHE enhanced myeloid and granulocyte counts whilereducing the fraction of early megakaryocytes. However, in CFU-MKcultures, sequence-independent effects of the employed S-BCHE oligoincreased the variability in colony counts. In contrast, the variabilityin CFU-GEMM colony counts was reduced under AS-BCHE treatment, togetherwith suppression of megakaryocytes. These observations confirmed andextended applicants' previous findings [Patinkin et al., 1990;Lapidot-Lifson et al., 1992; Lev-Lehman et al., 1994; Ehrlich et al.,1994] while demonstrating that the hematopoietic diversion induced byAS-BCHE from megakaryopoietic toward the myeloidocenic lineages iserythropoietin-independent, involves increases in myeloid proliferationand occurs also under in vivo conditions. These findings also indicatethat CFU-GEMM progenitors respond to AS-BCHE in a less variable mannerthan CFU-MK progenitors. Individual progenitor cells may therefore beexpected to respond to specific AS-ODN agents with different levels ofvariability, dependent both on the oligo and on the cell type inquestion.

Similar to the effects of AS-ACRE, the suppression ofmegakaryocytopoiesis by AS-BCHE occurred throughout the dose-responsecurve of CFU-GEMM.

The long-term in vivo-ex vivo duration of AS-BCHE effects is of specialinterest. It indicates that the AS-BCHE-induced destruction of BCHEmRNAin young promegakaryocytes was capable of reducing development of thesecells for at least two weeks and demonstrates that no feedback responsesoccurred to compensate for BCHE suppression and retrieve normalproduction of megakaryocytes.

In general, the AS-BCHE effects were limited as compared with thedistinct effects caused by ex-vivo and in vivo treatment with theparallel AS-ACHE ODNs blocking ACHE expression. Like AS-BCHE, AS-ACHEalso suppresses megakaryocyte formation. However, unlike AS-BCHE, italso suppresses erythropoiesis ex-vivo and in vivo [Lev-Lehman et al.,1994; Soreq et al., 1994], suggesting that acetylcholinesteraseparticipates in the erythropoietic process as well. Moreover, AS-ACHE,but not AS-BCHE induces a dramatic ex vivo expansion of CFU-GEMM colonyproduction and cell proliferation and reduces apoptosis in CFU-GEMMprimary bone marrow cultures [Soreq et al., 1994]. These differencesreveal distinctions between the role(s) played by the twocholinesterases in mammalian hematopoiesis. Development of both novelanticholinesterases and AS-ODN agents targeted to these mRNAs as setforth in the present application take into consideration thehematopoietic involvement of the protein products of these mRNAs as wellas their distinct role in the hematopoietic process.

Example 3 AS-ODN in Mice at the Neuromuscular Junction

Another prominent site for ChE activities is the neuromuscular junction,where ChEs control the cholinergic innervation of motor functioning.Therefore, it would be important to ensure that only the desired tissuewill be affected under systemic administration of a specific AS-ODN.

In vivo administration of an AS-ACHE oligo altered hematopoiesis ininjected mice [Lev-Lehman et al., 1994] as described in Examples 1 and 2herein above. In order to apply this technology to an extended in vivouse, applicants asked whether injection of certain AS-ODNs always affectthe target mRNA in other tissues as well (FIG. 4).

Five week old, female, white Sabra mice were injected (i.p.) once perday for 3 days with 0.2 ml PBS or with PBS containing 3'-terminallyphosphorothioated oligodeoxynucleotides (5 μg/g body weight) targetedagainst the mouse ACHE gene. Two antisense (AS) oligonucleotides wereused, one targeted against the common exon E2 (mE2; mouse E2; SEQ IDNo:8) or the alternative hematopoietic exon E5 (mE5; mouse E5; SEQ IDNo:9) compared with those of sense (S) oligos based on the homologoushuman ACHE gene sequence or sham injections with PBS. β-actin mRNAserved as a control for non-specific effects on transcription. Mice weresacrificed 24 hours following the last injection and total RNA preparedfrom muscle and bone marrow (BM). Semi-quantitative RT-PCR was performedon 100 ng samples of RNA using a primer pair (+1361/-1844) anchored inmouse ACHE gene exons E4 (+) and E6 (-). Samples were removed foranalysis every 3 cycles between cycles 24 and 33. Both AS-mE2 and AS-mE5exert specific reduction of E6-containing ACHEmRNA in bone marrow butnot muscle at the administered doses while actin mRNA was unaffected byany treatment.

The AS-mE2 ODN potentially hybridizes to the three alternative splicingforms of ACHEmRNA transcripts that encode polypeptides differing intheir C-terminal peptide sequences (FIG. 4A): the "synaptic form"containing exons E2-E3-E4-E6, the "readthrough form" (containing exonsE2-E3-E4-I4-E5 and the "hematopoietic form" containing exonsE2-E3-E4-E5. AS-mE2 (an antisense sequence selected in the E2 exon) wastherefore expected to be highly efficient in all of the tissues whereAChE is expressed. On the other hand, AS-mE5 (an antisense sequenceselected in the E5 exon, SEQ ID No:9) should only be able to hybridizeto the last two forms, which limits its potential efficacy in the CNS.

RNA-based PCR amplification (RT-PCR) was performed on RNA extracted frombone marrow (BM), muscle and brain of the injected animals withdifferent PCR primers. To test whether these AS-ODNs exert non-specifictoxic effects on total RNA degradation RT-PCR with primers for β-actin(for primer sequences see Lev-Lehman et al., 1994) was employed. Aneffective decrease in the level of E6-ACHE occurred in BM but not brainafter AS-mE2 ODN injection as compared with a subtle decrease in muscle(FIG. 4B). A more limited decrease in E6-ACHE mRNA was observed inmuscle and bone marrow, hut not brain, of animals treated with theAS-mE5 ODN. This could reflect limitations in access into the brain aswell as hybridization with the primary transcript of AChEmRNA in thenucleus of the muscle and cells of the bone marrow, leading to itsdegradation or inhibition of the splicing process and transport into thecytoplasm. These results are in agreement with the already discussedhigher susceptibility of bone marrow to AS-ODN. Thus, in vivoadministration of AS-ODN does not necessarily cause the same effect indifferent tissues expressing the targeted proteins. This allows thedesign of specific AS-ACHE-ODNs to be targeted to specific tissues.

Example 4 In Vitro Testing of AS-ODNs

The PC12 cell line, derived from rat phaechromocytoma cells, is awell-established model for studying vertebrate cholinergic neurons whichcan be induced to differentiate by nerve growth factor (NGF). NGFtreatment is shown to arrest the proliferation of PC12 cells, changetheir gene expression pattern [Lee et al., 1995] and induce theirdifferentiation toward a cholinergic phenotype with increased AChEactivity and neurite-like processes [Greene and Tischler, 1976;Tao-Chang et al., 1995]. Therefore, NGF-pre-treated PC12 cells willdiffer significantly from non-treated ones in their membrane properties,cytoarchitecture and levels of ACHEmRNA.

These cells can be used as a model to screen for the neurotoxicity ofAS-ODNs which have been protected against nucleolytic degradation and todetermine if there is differences in responses depending on the stage ofdifferentiation of the cells. The series of AS-ACHE ODNs was tested onPC12 cells before, during and after induction of differentiation by NGF.

Materials and Methods

Cell Lines

Rat phaeochromocytoma PC12 cells were provided by Dr. R. Stein, Tel-AvivUniversity. Cells are grown in Dulbecco's modified Eagle's mediumsupplemented with 8% fetal calf serum, 8% horse serum, 2 mM glutamine,100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells are kept at 37° C.in a fully humidified atmosphere at 5% carbon dioxide. Fordifferentiation, 50 ng/ml NGF (Alomone Laboratories, Jerusalem, Israel)is added. All tissue culture reagents are from Biological Industries(Beit Haemek, Israel).

Primary Cultures

Primary mouse neuronal cultures are prepared from embryonic (e14) mouse(Balb/C) whole brains. Brains are removed and cells mechanicallydissociated by being drawn through a Pasteur pipette. Cells are platedin serum-free medium (2.5×10⁶ cells/ml) in 24-well (1 ml per well)Costar (Cambridge, Mass.) culture dishes coated successively withpoly-L-ornithine and culture medium containing 10% fetal calf serum[Weiss et al., 1986] Wherever mentioned, Actinomycin D is added for 72hours at 0.5 μg/ml.

Oligonucleotides

The AS-ODNS were synthesized by Microsynth (Balgach, Switzerland). TheODNs were 20 nucleotides in length with the last three 3'internucleotidic linages phosphorothioated. The seven ODNs tested weretargeted towards various sites along the mouse ACHEmRNA chain takinginto account exon splice variables (SEQ ID Nos:8-14). The most abundantmature transcript in brain is one in which exon 4 is spliced to exon 6.The mAS-ODNs had the following sequences:

    Experimental Sequences:                                                            mAS1  5'-GGGAGAGGAGGAGGAAGAGG-3'                                                                         SEQ ID No:8                                     (ASmE2)                                                                       mAS2           5'-TAGCATCCAACACTCCTGAC-3' SEQ ID No:10                        mAS3           5'-CTGCAATATTTTCTTGCACC-3' SEQ ID No:11                        mAS4           5'-ATGAACTCGATTTCATAGCC-3' SEQ ID No:12                        mAS5   5'-AGAGGAGGGACAGGGCTAAG-3' SEQ ID No:9                                 (ASmE5)                                                                       mAS6                     5'-GTCGTATTATATCCCAGCCC-3' SEQ ID No:13                                             mAS7         5'-GTGGCTGTAACAGTTTATTG-3'                                      SEQ ID No:14                                  Control Sequences                                                                MASB     5'-GACTTTGCTATGCAT-3'                                                                              SEQID No:15                                    mI-AS5 5'-GAATCGGGACAGGGAGGAGA-3'  SEQ ID No:16                         

mAS1 (position in neuronal mouse transcript 70) and mAS2 (880) are inclose proximity to the translation initiation site in exon 2. mAS3 (658)and mAS4 (1454) are located in exons 2 and 3 common to all the splicevariables. mAS5 (234) is targeted to exon 5; this particular ODN shouldhybridize with the alternative E5 ACHEmRNA, yet not with mature E6transcript. mAS6 (1932; SEQ ID No:13) and mAS7 (2068; SEQ ID No:17) weredesigned to hybridize with exon 6. No mAS-ODN was designed for I4, sinceits sequence is the most variable among mammals [Karpel et al., 1994].All mAS-ODNs, except mAS6 and mAS7, were targeted againsttranslationable sequences included in the open reading frame ofACHEmRNA. (see FIG. 5B for schematic position of AS-ODN in gene)

Antisense Treatment

PC12 cells are grown to 50% confluence (approx. 105 cells per well) in96-well Nunclon™ (Nunc, Roskilde, Denmark) microtiter plates. Following24 hours in culture, fetal calf and horse sera are reduced to 2% eachand either 1 or 10 μM ODN added to the culture medium for an additional24 hours. In certain experiments, Lipofectamine™ was added together withthe ODN essentially as instructed by the producer (GicoBRL,Gaithersburg, Md.), except that 1 μM ODN is used together with 2.5μlLipofectamine™ per well.

Colorimetric Measurements

Following ODN treatment, cells are washed once with phosphate-bufferedsaline and lysed with 1% Titon X-100 in 200 μl of 100 mM phosphatebuffer, pH 7.4 containing 0.5 mM dithio-bis-nitrobenzoic acid (DTNB) for20 minutes. Washing removes dead cells, which do not adhere to the wellsurface. To evaluate cell survival after AS-ODN treatment, the contentof free thiol groups in these cells is measured. Such groups react withDTNB to yield the yellow anion 5-thio-2-nitrobenzoate, which can bequantified in the same microtiter wells by absorption at 405 nm (ε₄₀₅=13,600 M⁻¹ cm⁻¹). Such absorbance was found to be proportional to theconcentration of cells within each well and served as a measure of cellnumber (FIG. 6A). AChE activity was subsequently measured following theaddition of 1 mM acetylthiocholine to the DTNB solution in the samewells, using an adaptation of Ellman's assay [Ellman et al., 1961] foruse with 96-well microtiter plates [Seidman et al., 1994]. For testingAS-ODN-AChE interactions, similar assays were performed with highlypurified recombinant human AChE (Sigma Chemical Co., St. Louis, Mo.,USA) incubated with the noted quantities of ODNs.

RNA Extraction and PCR

Total RNA was extracted from whole brain, embryonic brain neurons andPC12 cells, using RNazol™ (Biotecx Laboratories, Inc., Houston, Tex.) asdetailed elsewhere [Karpel et al., 1994]. Reverse transcription followedby PCR amplification was performed as described by Karpel et al., 1994.

Kinetics of accumulation of RT-PCR products is studied by removal of 12μl aliquots at 6 alternate cycles in the PCR procedure. Collected DNA iselectrophoresed on ethidium bromide stained agarose gels. UV images ofthese gels are digitized using a charge coupled device (CCD) camera. Theintensity of fluorescence is quantified using the program IpLab Spectrum(Signal Analytics, Vienna, Va., USA), for quadruple PCR reactions.Resultant values are plotted as percent of the maximal intensityobtained at a time point when the control set of PCR reactions reaches aplateau. Under ideal conditions, fluorescence intensity should increaseexponentially throughout this kinetic follow-up, with the verticalseparation between individual curves dependent on the initial quantityof the examined mRNA. Linear regression analysis of relativefluorescence units vs. Cycle number should therefore yield an estimateof the amount of the template originally present. In cases whereselective mRNA destruction took place, the levels of the target mRNA,but not an irrelevant control mRNA should show vertical shifts in thekinetic accumulation curves, reflected in different intercepts with they axis.

Results

The three alternative ACHEmRNA splice variants are present in P12 cellswith E6>I4>E5 (FIG. 5A), a pattern similar to that found in bothembryonic mouse brain neurons and adult mouse brain.

In the experiments reported herein, the AS-ODNs were protected by3'-phosphorothioation. Since the original ACHE transcript may bealternatively spliced to produce three different mRNAs, in this studythe efficacy of AS-ODNs targeted the different mature mRNA isoforms insuppression of the production of AChE in differentiated (NGF-treated)and non-differentiated cells was undertaken.

Three different administration protocols were used: non-differentiatedPC12 cells were treated with 1 μM AS-ODN alone or with NGF for 24 hours,or NGF-induced differentiation was allowed to proceed for 24 hoursbefore being exposed for a second 24 hours to the AS-ODN.

To evaluate neurotoxicity, the number of live cells was determinedaccording to the content of free thiol groups in in situ lysed cells.The rate of acetylthiocholine hydrolysis was the measure of AChEactivity. The effects of each ODN on cell survival were studied byquantitating the reactive free thiol groups in Triton X-100-lysed cellsas a measure of cell number. This measurement was fast, convenient andsimple to perform; a linear relationship was found between the number ofcells plated in individual wells and the content of free thiol groups inthe culture (FIG. 6A). A similar relationship was observed forNGF-treated cells. A reduction of >20% in free thiol groups was taken asan indication of toxicity. At a concentration of 1 μM, none of the ODNsreduced the content of free thiol groups in the cultures by more than5%, except for mAS2. Some toxicity was, however, observed at aconcentration of 10 μM, where 5 out of the 9 ODNs (Nos. 1, 3, 5, 6 and7) reduced the content of free thiol groups by 20-40% (FIG. 6B).

To facilitate the uptake of the ODNs into PC 12 cells, we testedreactive liposomes (Lipofectamine™). Under these experimentalconditions, Lipofectamine™ seemed to be extremely toxic to the cells,especially after differentiation, and reduced their number to as low as10% within 24 hours. Therefore, its use was discontinued.

The capacity of these ODNs to suppress AChE activity was testedseparately in three sets of growth conditions; (1) for cells in theabsence of NGF, (2) for co-administration of AS-ODNs and NGF, and (3)following 24 hour differentiation with NGF, Table III presents theefficacy of each of the tested ODNs in suppression of AChE activity invarious PC12 cultures.

AChE activities in control ODN-treated non-differentiated cells werelower than those in non-treated cells by 9 and 10%. One out of the 7AS-ODNs, mAS3, suppressed AChE activity in non-differentiated PC12 cellsby over 20% (P≦0.01, Student's t-test) (Table III, column A). Asexpected, an increase of approximately 13% in AChE specific activity wasobserved 24 hours after addition of NGF, so that acetylthiocholinehydrolysis levels increased from 7.8 to 9.0 nmol/min/10³ cells underthese conditions. Co-administration of AS-ODNs with NGF resulted invariable yet apparently effective (12-28%) suppression; however, 16%inhibition was observed also in cells treated with the control ODNs.This, and the large variability between inhibition values in differentcultures, indicated that much of the effect of AS-ODNs was primarilysequence-independent under these conditions.

Only one AS-ODN, mAS5, exerted significant (28%, p≦0.01), more thantwo-fold control inhibition under co-treatment conditions (Table III,column B). Twenty-four hours later, AChE activity increased further to11.7 nmol/min/10³ cells. Assuming 10⁶ cells per mg wet weight and 10%protein, this is equivalent to 1.2 μmol/min/mg protein, which isconsiderably higher than the specific activity of 0.22 μmol/min/mgprotein for homogenates of mouse brain cortex found by Berri et al.[1995]. Interestingly, a significant part of this increase was preventedwhen AS-ODNs were added to cells that had been pre-treated with NGF for24 hours. In these cells, yet two other AS-ODNs, mAS1 and mAS4,suppressed AChE activities by over 25% and 36%, respectively, ascompared with a limited suppression (up to 11%) by control ODNs(p≦0.01).

mAS3, effective in non-differentiated PC12 cells, and mAS5, effectiveunder co-administration of NGF and AS-ODN, inhibited 21 and 20% of AChEactivity in NGF pre-treated cells, respectively (Table III, column C).Of these, mAS3 was more significantly effective than mAS5 (p≦0.01vs.≦0.05).

FIG. 7 presents the efficacy of each of the AS-ODNs as a function of theposition of its target sequence along the ACHEmRNA chain. No patternrelating the sequence position to which an AS-ODN was targeted wasdetected within the ACHEmRNA chain and its efficacy in suppressing AChEactivity, either in non-differentiated or in NGF-pretreated cells.Inactive ODNs included the apparently toxic mAS2 ODN, which did notsuppress AChE activity at all, and the 3'-terminal AS-ODN targeted to E6(mAS-7), which was relatively inefficient under all three growthconditions. Interestingly, mAS5, which was effective in co-treated cells(Table III, column B) and in primary cultured differentiating mouseneurons [Grifman et al., 1997], was relatively inefficacious innon-differentiated PC12 cells. mAS4, which suppressed AChE activity by36% in NGF pre-treated cells, was rather ineffective both innon-differentiated cells and under co-administration conditions.

To test the possibility that the inhibition of AChE activity in AS-ODNtreated cells was due to aptamer effects of the tested oligos on thecatalytic activity of the enzyme, the purified recombinant human AChEwas incubated for 24 hours in phosphate buffered saline (PBS) including1% bovine serum albumin and 1 μM of the relevant AS-ODNs. Subsequentmeasurement of catalytic activates as compared to those of AChEpreparations incubated in PBS alone demonstrated that mAS1, mAS3, mAS4,mAS5, and mAS6 did not modify the catalytic activity by more than 3%.

To obtain an independent measure of the inhibition of AChE expression,total RNA was extracted from PC12 cells which were pre-incubated for 24hours with NGF and then for 24 hours with either mAS1, mAS3, mAS4, mAS6,a control ODN (AS-B) or no ODN. The levels of AChE mRNA in these cellswere evaluated by a kinetic follow-up of reverse transcription coupledto PCR amplification (FIG. 8). This semi-quantitative analysis clearlyrevealed similar kinetics (parallel lines in the accumulation plots) aswell as a decrease in AChE mRNA levels in AS-ODNs-treated cells but notin control cells or in those treated with the control ODN (reflected bya shift to the right in the accumulation curve). Moreover, actin mRNAlevels, when subjected to the same analysis, remained unchanged in allof these cell cultures, demonstrating the selectivity of ACHEmRNAreduction under the effective AS-ACHE ODNs.

In summary, two out of seven AS-ODNs designed to hybridize with ratACHEmRNA (mAS1 and mAS4) suppressed AChE activity in PC12 cells thatwere pre-treated with NGF by over 25%, while leaving cell numbersunaffected. Neither of these was effective in non-differentiated PC12cells or in NGF co-treated cells, where they did not suppress AChEactivity significantly more than the control ODNs. These two ODNs targetexons that are common to all the alternatively-spliced forms ofACHEmRNA, a positioning factor which may be relevant to their highefficacies. In contrast, the limited secondary structure predicted bytheoretical considerations for mAS3 and mAS4 (.increment.G:=-4.7 and-2.6 Kcal/mol, respectively) or their low G,C content (40%), seem to beof no significance to their antisense efficacy, as other AS-ODN agentswith similar properties (e.g. AS7) were considerably less effective. TheFOLDRNA program (University of Wisconsin GCG software package) revealsthat mAS4 targets a region with a relatively loose predicted stem-loopcomposition (not shown). However, mAS1, also effective in NGFpre-treated cells, targets a tightly folded stem region. In addition, itis not apparent that the structure drawn for this 2.3 Kb and mRNA isbiologically significant. Thus, none of the standard physical parametersused to characterize AS-ODNs explains the apparent superiority of mAS1and mAS4 compared to the other AS-ODNs.

An intriguing implication of the Example is that neurons might beconsiderably more susceptible to AS-ODN inhibition than theirundifferentiated precursors. This property may reflect relativelyefficient uptake of ODNs, enhanced activity of neuronal RNaseH, moredeveloped vulnerability to bona fide AS mechanism(s), or a combinationof all three. The first two possibilities are less likely, since thecontrol ODN was similarly inactive in PC12 cells that had or had notbeen pre-treated with NGF. This suggested no difference in ODN uptake orin non-specific RNaseH activity. The third option, in turn, indicatesdistinct mechanisms for specific AS-ODNs functioning in neurons atvarious stages of differentiation. This option is strengthened by thefinding that mAS4 was the most effective in NGF-treated PC12 cellswhereas mAS3 was the most effective in undifferentiated PC12 cells. Thelikelihood of an AS mechanism(s) is further supported by the effectiveAS-ODN suppression in NGF-stimulated PC12 cells, in spite of the factthat AChE levels increased significantly in such cells. This may be dueto enhanced translation, which may increase the susceptibility of AChEmRNA to AS-ODN-mediated destruction. Increased stability of ACHEmRNA indifferentiated neurons, as compared with their progenitors, should alsobe considered, as this was shown in the P19 embryonal neuron cell line[Coleman and Taylor, 1996] and corroborated. However, ACHEmRNA may indifferentiated neurons be less protected by cellular protein(s) againstRNAse H attack as compared with the less active ACHEmRNA innon-differentiated neurons. Finally, the apparent inhibition of AChEaccumulation in NGF pre-treated neurons may reflect a faster turnover ofthe active enzyme in these cells. Therefore, AS-ODN may be moreefficient in NGF-treated neurons due to antisense mechanism(s) supportedby potentially enhanced AChE production and faster turnover in thesecells, and in spite of the slower turnover of ACHEmRNA in differentiatedneurons.

mAS7, targeted to the 3'-region of exon 6, was significantly lesseffective than those designed against the sequence common to allalternatively-spliced ACHEmRNA transcripts. This was the case in theabsence of NGF, under co-treatment conditions and following 24 hourtreatment with this differentiation inducing agent. This is not ageneral rule; on the contrary, AS-ODNs against 3'-regions in other mRNAswere shown to effectively induce destruction of the entire mRNA sequence[e.g. Bennet et al., 1994]. Indeed, a methodical study by Falkler et al.[1994] demonstrated efficacy of ODNs, unrelated to the location of theirtarget sequence in the mRNA. However, mammalian ACHEmRNA is especiallyrich in G,C base pairs (67% in human ACHE, Soreq et al., 1990).Therefore, it is likely to be tightly folded. Since a truncated humanACHEmRNA bearing only exons 2, 3 and 4 was found to be translatable inXenopus embryos [Seidman et al., 1997], it is possible that E6-ACHEmRNAis so tightly folded that RnaseH action on its 3'-exon does not lead todestruction of exons 2, 3 and 4, leaving an mRNA which encodes acatalytically active, C-terminally truncated protein.

These findings demonstrate a specificity of several of the AS-ODNs, bothfor differentiated neurons as target cells and for ACHE expression,showing that specific AS-ODNs can be used to suppress AChE levels in thetreatment of diseases associated with cholinergic malfunction ordiseases requiring control of cholinergic expression.

Example 5 Testing of AS-ODNs in Transgenic Mice

AS-ACHE-ODNs have been produced and injected which are targeted againstboth human and mouse AChEmRNA (see Tables I and II). AS-ODNs wereprotected by one of two modifications: a) phosphorothioate modificationof the last three nucleotides (3' phosphorothioated) or b) 3' additionof a 9 base palindromic sequence (SEQ ID No:3) designed to create anuclease resistant loop (3' looped). The scientific basis for thesemodifications is presented by Ehrlich et al. [1994].

Materials and Methods

Enzyme Activity Assays

Cerebral hemispheres were dissected into cortical and subcorticalregions, frozen in liquid nitrogen and stored at -70° C. until used. ForAChE activity measurements, extracts were prepared in 10 vol. (wt/vol)10 mM phosphate buffer containing 1% Triton-X 100 using a glass-glasshomogenizer, incubated on ice for 1 hour and microfuged in the cold for30 minutes. Cleared homogenates were diluted 1:10 and 10 μl assayed in200 μl final volume 0.1 M phosphate buffer (pH 7.4), 0.5 mMdithiobis-nitrobenzoic acid, 0.1 mM acetylthiocholine. Proteindeterminations were performed using a commercial assay kit (Promega).Enzyme-antigen immunoassay was performed using a species-specificmonoclonal antibody (mAb 101-1) to identify AChE of human origin inhomogenates.

RNA Extraction

Isolation of RNA was made by the RNA-Clean™ method (AngewandteGentechnologic Systeme GmbH, Heidelberg, Germany). Samples werehomogenized in 0.8 ml RNA-Clean and transferred to Eppendorf tubes. 80μl chloroform was added to the homogenates and stored for 5 minutes at4° C. Samples were then centrifuged for 15 minutes and the aqueous phasewas collected into new Eppendorf tubes. 0.4 ml of isopropanol was addedfor 45 minutes at 40° C. RNA precipitates were later centrifuged for 15minutes and washed once with 0.8 ml of 70% ethanol.

RT-PCR Amplification

RT-PCR was performed essentially as described [Beeri et al., 1995] usingspecific primers for human AChE and mouse AChE, CHAT, actin, andsynaptophysin. Cycling reactions were performed at 69° C. RT-PCR wasperformed in a thermal cycler (GeneAmp PCR System 9600, Perkin-ElmerCetus Corp. South San Francisco, Calif.). Each tube contained a finalvolume of 10 μl, consisting of 2 μl RNA sample, 3 μl DDW, 1 μl dNTPs (4mM), 0.5 μl hexamers (2.5 μM), 2 μl 5 X PCR buffer, 0.25 μl HPRI, 1 μlDDT (100 mM) and 0.25 μl RT enzyme. After 40 minutes at 37 ° C., 40 μlof PCR reagents were added, so that total volume in the tubes was 50 μl.PCR reagents consisted of 4 μl 10 X PCR buffer 30.75 μl DDW, 2.5 μlprimer (+, 10 μM), 2.5 μl primer (-, 10μM) and 0.25 μl of Taq DNApolymerase. Resultant PCR products were electrophoresed on 1.5% agarosegels and visualized under UV illumination following staining withethidium bromide.

In Vivo Injections

Protocols for delivering antisense oligonucleotides to transgenic micein vivo by intravenous (i.v.; tail vein), intraperitoneal (i.p), andintracerebroventricular (i.c.v) routes were developed. To test thevalidity of these various administration routes, 12-15 day-old mice wereused that can be later used to test early prevention schemes.

i.v.

12-week-old ACHE transgenic mice were placed briefly under a warminglamp, injected into the tail vein with 5 μg/gr body wt. oligonucleotidein a volume of 0.1 ml in PBS, and sacrificed 18 hours later bydecapitation.

i.p.

Mice were injected intraperitoneally with 5 μg/gr body wtoligonucleotide (0.5 mg/ml). Both single injection and multipleinjection protocols were explored. For multiple injections, animals wereinjected at 24 hour intervals for 3 days. Mice were sacrificed 18 hoursfollowing last injection.

i.c.v.

10-12 day old ACHE transgenic mice were injected i.c.v. into the leftlateral ventricle with 0.2-0.4 μl oligonucleotide (50-200 μM) in PBScontaining Evans blue as a marker for monitoring accuracy of theinjections. For surgery, animals were anesthetized with ether and asmall incision was made in the scalp. A small hole was made with a 25gauge hypodermic needle and injections were performed using a 10 μlHamilton syringe. Mice were returned to the mother following a 1-2 hourrecovery period and sacrificed 18-40 hours post-injection bydecapitation. Brains were excised and cerebellum discarded.

Results

Six experiments involving in vivo injections into live animals asdescribed in Table II were performed.

RNA (200 ng) from cortex of mice injected i.v. with buffer or with ASoligodeoxynucleotides targeted against hACHE (AS1120, SEQ ID No:1;AS1500, SEQ ID No:2) or mACHE (ASmE2, SEQ ID No:8) were subjected tosemi quantitative kinetic follow-up of RT-PCR amplification products asdescribed in herein above. Specific primers were employed to detecthACHE, mACHE or synaptophysin (Syn) mRNAs. cDNA product was collectedevery third cycle between cycles 21-36, subjected to gel electrophoresisand stained with ethidium bromide. The products from cycles 21-36 arepresented in FIG. 2 from left to right. First appearance of cDNA productand/or intensity of bands were taken as measures of original mRNAconcentration. For hACHE note the lower intensity of the first two bands(cycles 27,30) in all antisense oligodeoxynucleotide treated micecompared to buffer injected control. For mACHE note that the firstappearance of product in the ASmE2 treated mouse is delayed by threecycles compared to both buffer injected and hAS injected mice. Thecontrol synaptophysin mRNA levels were identical in all samplesindicating that an approximately equal amount of RNA was introduced intoeach PCR reaction and that AS-ODNs did not cause non-sequence dependentcellular toxicity.

Levels of AChE activity in cortex of mice injected with buffer or withAS oligodeoxynucleotides are presented in nmol substratehydrolyzed/min/ug protein in chart in FIG. 2. There is a decline in AChEactivity in the cortex of the two mice injected with AS1500. As shown inFIG. 3, antisense oligonucleotides injected i.c.v. give a reduction inAChE catalytic activity in subcortical regions.

No acute toxic effects were observed in any AS-ODN treated humantransgenic mouse and behavior appeared normal in all treated animals. Invivo experiments were performed on littermates only. AS-ODN targetedagainst hAChEmRNA resulted in diminished levels of both hAChE- and andmAChE mRNAs (FIG. 2) and dramatically reduced protein levels in one oftwo animals. AS-ODN against mAChEmRNA resulted in a 3 cycle delay inappearance of RT-PCR product in one animal (approx 8-fold reduction inmRNA). When 100 pmole (approx 1 ug) AS-ODN against hAChE- or mAChE- mRNAwas delivered i.c.v. to 15 day old mice, 2 of 3 mice in each groupdisplayed total AChE activities >1 S.D. below the mean activity measuredin buffer injected animals 40 hours post injection (FIG. 3).

The above results in combination with Examples 7 and 8 herein belowdemonstrate that the human transgenic mouse model provides a model fortesting human AS-ACHE-ODNs for efficacy.

Example 6

Cortico-Hippocampal Brain Slices are Useful as an Ex Vivo System forEvaluating Anti-ACHE-ODNs Efficiency in Mammalian Brain

For the first stage in the development of antisense (AS)oligodeoxynucleotide (ODN) therapies directed against the human ACHEgene in brain, it is essential to have a rapid and convenient model forscreening candidate ODNs in a heterogeneous population of cells of themammalian central nervous system (CNS). To this end, applicantsestablished an assay system utilizing cortico-hippocampal brain slicesfrom mice, including transgenic mice carrying the human ACHE gene,together with electrophysiological, biochemical, and molecular analyses.

In this assay 400 μM murine brain slices can be maintained in vitro forat least 11 hours after which intact, PCR-amplifiable RNA andcatalytically active AChE protein may be prepared. Moreover, brainslices are amenable to cytohistochemical analyses including in situhybridization, cytochemical activity and immunohistochemical staining todetermine the precise localization of AChE mRNA and protein expressionin various brain regions. Using this system, applicants havedemonstrated that application of various acetylcholinesterase (AChE)inhibitors including tacrine (THA, tetrahydroamino-acridine,Cognex®)--the first FDA-approved Alzheimer's disease (AD) drug--induce a2-fold increase in AChE activity that is preceded by enhanced levels ofa specific AChE-encoding messenger RNA. This elevation in AChE activitywas associated with enhanced neuronal excitability and is accompanied bychanges in the expression of additional genes important in neuronalactivity.

Thus, in comparison to cell culture systems, the cortico-hippocampalbrain slice system offers a convenient in vitro model to examine theefficacy and mode of action of antisense oligonucleotides targetedagainst AChEmRNA on primary CNS neurons in the context of their naturalsurrounding tissues while maintaining many native cholinergic signalingpathways at least partially intact. The main advantage of this approachover in vivo studies is that it overcomes the technical limitationsimposed by the blood-brain-barrier by facilitating direct access tobrain tissue for the administration of drugs. Moreover, it allows formultiple experimental analyses to be performed on tissues extracted froma single mouse, dramatically reducing the number of animals sacrificedfor this research.

Method

For preparation of brain slices, mice were anesthetized with nembutal(60 mg/kg) and decapitated. Brains were removed into ice cold NSR buffer(124 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.25 mM NaH₂ PO₄, 26 mM NaHCO₃, 10mM D-glucose, 2 mM CaCl₂ ; pH 7.4) Continuously aerated with 95% O₂ /5%CO₂. Vibrotome sections (400 μm) were prepared and maintained in aeratedNSR buffer at room temperature. Slices were allowed to rest undisturbedat least 1 hour before any additional manipulations were performed.Slices were transferred to individually aerated bottles allowing atleast 2.5 ml buffer per 2 slices and the concentration of KCl raised to8 mM to hyperpolarize the cells prior to the addition of inhibitors.

Results

Transcriptionally regulated shutoff of cholinergic neurotransmissionfollowirg cholinergic hyperactivation: During acute stress reactioncentral cholinergic pathways are fully activated. To explore themolecular consequences of cholinergic hyperactivation, we subjectednormal FVB/N mice to a forced swimming stress protocol or exposedcortico-hippocampel brain slices to cholinesterase inhibitors andsearched for accompanying changes in brain gene expression. Both stressin vivo and AChE inhibition in vitro stimulated rapid and specificincreases in "readthrough" AChEmRNA encoding a soluble hydrophilic AChEwith potentially greater intercellular accessibility than the classicsynaptic form of the enzyme.

In situ hybridization revealed "readthrough" AChEmRNA transcripts incell bodies and apical processes of pyramidal neurons within corticallayers 2, 3, 4 and 5 in brain sections from mice injected with theanti-AChE pyridostigmine, as compared with weaker, more restrictedlabeling in cell bodies located in layer 2 and layer 5 neurons fromcontrols. Increased AChEmRNA levels induced up to 3-fold enhanced levelsof catalytically active enzyme in hippocampus and cortex but not incerebellum within 5 hours. Stress-enhanced AChE activity wascharacterized by increased heterogeneity and overall faster migration innon-denaturing gel electrophoresis. In contrast, both stress andinhibition of AChE stimulated pronounced reductions in ChATmRNA levels,suggesting that a bimodal mechanism comprised of suppressedacetylcholine synthesis and enhanced acetylcholine hydrolysis works toshut down cholinergic neurotransmission following acute hyperactivation.Although both treatments resulted in increased c-fos mRNA levelsindicating neuronal excitability, no changes were observed insynaptophysin mRNA levels, demonstrating the selectivity of this"cholinergic" feedback response. In brain slices treated with AChEinhibitors increased neuronal excitability, paired-pulse facilitation,and mRNA changes were blocked by both BAPTA-AM and tetrodotoxin,indicating that these processes are mediated by increases inintracellular Ca++ and/or Na+ influx.

These experiments demonstrate the utility of the brain slice system inmonitoring changes in ACHE gene expression and the utility of ACHEtransgenic mice as a novel model for studying the efficacy ofAS-ACHE-ODNs.

Tacrine-Induced Elevation of AChE Expression

Tacrine is a potent reversible AChE inhibitor which relieves cognitivesymptoms in 30-50% of mildly to moderately affected AD patients. Theobservation that irreversible inhibitors such as DFP or pyridostigmineinduce lasting changes in the expression of genes relating tocholinergic pathways, including feedback pathways elevating AChE levels,suggested that tacrine may induce similar responses. To examine thispossibility, tacrine was applied at a concentration of 5 ×10⁻⁷ M tobrain slices for 75-90 minutes and examined AChE activity in detergentextracts. Under these conditions, AChE activities of 26-186% above thosemeasured in control untreated slices were observed.

These observations reinforce the utility of cortico-hippocampal brainslices in the study of AChE gene expression and provide for the use oftacrine in studies of the efficacy of antisense oligonucleotidestargeted against AChEmRNA in suppressing AChE biosynthesis in asensitive, short time-frame model. Moreover, they emphasize theimportance of finding alternatives to the current cholinesteraseinhibitor approach to treating AD.

Example 7

Deficient Performance of HaCHE Transgenic Mice in Memory Tests Based onSocial Exploration or Taste

Social Exploration

Applicants previously demonstrated impaired performance of transgenicFVB/N mice expressing human acetylcholinesterase (AChE) in cholinergicbrain neurons in the Morris water maze for spatial learning and memory[Beeri et al., 1995]. Although one-month-old transgenic mice performsimilarly to control mice, progressive deterioration in the performanceof transgenic mice is observed to the age of 6-8 months at which pointthey have difficulty performing the task altogether. Together withneuropathological analyses [Beeri et al., submitted], these findingsappear to depict a chronic cholinergic imbalance leading to late-onset,progressive cognitive deficiencies--a novel model for the cholinergicimpairments associated with Alzheimer's disease. However, recent studiesrevealed severe visual impairments in AChE transgenic mice from aroundtwo weeks of age. Since performance in the Morris water maze purportedlydepends primarily on visual clues, it became important to conductadditional studies using a learning/memory paradigm that does notrequire intact visual networks to validate the model.

The experimental approach: To study the progressive cognitive deficitsobserved in AChE transgenic mice by an approach independent of visualfunctioning, the behavior of these mice in a test of social explorationwas observed. The test includes exposure of an adult mouse, eithertransgenic or control, to an unknown juvenile. This initiates anolfactory response of sniffing which lasts approximately 240 seconds.When the young mouse is removed and then immediately presented again(second presentation), the sniffing period shortens to about 80 seconds.This is a test of working memory and takes place similarly intransgenics and controls. When a different young mouse is substitutedfor the second presentation, it will be sniffed ca. 200 seconds,indicating a clear distinction between exploration of "same" and"different".

Ten minutes later, an adult control mouse will need 150 seconds toascertain recognition. After 20 minutes it will need 200 seconds andafter 30 minutes it will repeat the whole ritual as if this same mousewas not known to it at all. In the case of the transgenic mice "same" istreated as "different" even after a lapse as brief as 10 minutes,demonstrating a clear deficiency in this behavior (FIG. 9A).

Effect of Tacrine

This short-term behavior is described in the literature as dependent oncholinergic pathways, and emphasizes that cholinesterase inhibitorsextend the explorative memory. Tacrine as shown in Example 6 inducedelevation of AChE expression and utilizing this test, the effect oftacrine on hAChE transgenic mice was tested. As shown in FIG. 9B i.p.injection of 1 mg/ml tacrine extended short-term memory to 20 minutes inyoung (6 week old) transgenic mice.

This Example provides additional data that hAChE-transgenic mice indeedsuffer from progressive cognitive deficits that can be traced tocholinergic malfunction(s) that respond, at least in part, toanticholinesterase therapy for some time. Further the social explorationtest offers a relatively simple, rapid test to examine the efficacy ofanticholinesterase therapies, including antisense oligonucleotidestargeted against human AChE mRNA.

Taste Aversion Memory

Unlike the social exploration test, the taste aversion is designed todistinguish between defects in learning and long-term memory. Groups ofnon-transgenic and transgenic mice (10 mice per group), controls weresaline-injected, were established. The mice were kept thirsty for 20hours and then allowed to drink saccharine-sweetened water. The micedrank a volume of 2 ml (about 10% of their body weight). Thirty minuteslater they are injected with a nausea-causing lithium chloride solution.After 3 days, they were tested for learning and short-term memory with asaccharine challenge. In general, the mice drank only 1.2 ml, that isthey learned and remembered that the sweet taste causes nausea. The micethen received a second conditioning with the lithium chloride. Up tothis point both the control and transgenic mice responded the same (FIG.10).

To test long term memory, once a week, for several weeks after theinitial challenge and conditioning, the mice were given saccharine water(but no lithium). Control mice continued to drink less for weeks showinglong term memory. However, the transgenics gradually forgot, returningto full volume drinking within less than a month (FIG. 10).

The reference memory function required to stabilize the taste aversionexperience depends on cholinergic function which is impaired in thetransgenic mice.

Example 8 Effect of AS-ODN on Transgenic Mice in the Social ExplorationTest

An evaluation of 2-O-methyl AS-ODNs targeted to AChEmRNA to promoteimproved performance of ACHE transgenic mice in the learning and memorytest of Social Exploration and to correlate changes in behavior withmodulations in AChE protein and/or RNA levels. The experiment wasrepeated twice with the same results. Following is the results from onerepresentitive experiment.

Methods

Cannulation

Cannula were stereotaxically implanted into a ventricle of 5-month oldHpACHE mice approximately 2 weeks prior to the experiment and housedsingly during the recovery period.

Oligonucleotides and In Vivo Administration

5 mM 2-O-methyl (last three 3' nucleotides) oligonucleotides targetedagainst murine AChE (ASmACHE3-Me; mAS3; SEQ ID No:11) or BuChE(ASmBCHE-Me; mASB) were combined with 13 mM of the lipophilictransfection reagent DOTAP (Boehringer Mannheim) in phosphate bufferedsaline and incubated for 15 minutes at 37° C. prior to first injection.1 μl(25 ng) ODN was infused icv over a period of 1-2 minutes, 2 times,at 24 hour intervals. Mice were subjected to the social exploration testand sacrificed 24 hours following the last administration of ODN.

Sequences

mAS3: 5'-CTGCAATATTTTCTTGCACC-3' (20 mer; SEQ ID No:11)

mASB: 5'-GACTTTGCTATGCAT-3' (15 mer; SEQ ID No:15)

Social Exploration Test

Mice were placed in a cage with a glass front panel together with anunknown juvenile mouse for 4 minutes and the times invested by the adultmouse in exploratory contact with the juvenile recorded by an observer.At the end of the "baseline" test, the juvenile was removed from thecage. Ten minutes later, the same juvenile was reintroduced to the cageand the exploratory time recorded ("test"). The ratio of test:base timedetermined the "performance ratio" (PR). PR<1 represents "learning andmemory".

Cannulated mice were evaluated in social exploration 24 hours prior tothe first administration of ODN (PR1) and 24 hours following the lastadministration (PR2). PR2:PR1 was taken as a measure ofoligonucleotide-induced improvements in learning and/or memory.

Biochemistry

Animals were sacrificed by cervical dislocation, brains removed;hippocampus and cortex were dissected from each hemisphere, frozen ondry ice, and stored at -70(C until used. Low-salt-detergent extractswere prepared in cold 0.1 M phosphate buffer (pH 7.4) containing 1%Triton X-100 (1:9 wt/vol), and assayed for AChE activity using acolorimetric assayed based on the hydrolysis of acetylthiocholine (1 mM)(see herein above, Ellman et al, 1961). Protein determination wasperformed using the detergent-compatible (DC) kit from Biorad which isbased on the assay of Lowry.

Results

Tables 4 (experimental) and 5 (control) present the results. TheAChE-transgenic mice which suffer from progressive cognitive deficits(see Example 7) that can be traced to cholinergic malfunction(s) thatrespond, at least in part, to anticholinesterase therapy. As can beseen, the AS-ODN of SEQ ID No:3 improved performance in the transgenicmice. An anti AChE-ASODN can promote improved learning following 48hours administration. ODN-mediated modulations of AChE proteins levelsare reversible within 24 hours of administration.

Throughout this application, various publications, including U.S.patents, are referenced by citation or number. Full citations for thepublications are listed below. The disclosures of these publications andpatents in their entireties are hereby incorporated by reference intothis application in order to more fully describe the state of the art towhich this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

                                      TABLE 1                                     __________________________________________________________________________    Antisense Oligonucleotides                                                                              SPE- PROTE                                            NAME     SEQUENCE                                                                                                                 CIES   CTION                                                                  POSITION    MW                                                                SEQ ID                  __________________________________________________________________________      ASmE2                                                                              5'GGGAGAGGAGGAGGAAGAGG3'                                                                         mouse                                                                              3' PS ×3                                                                     51           6994 SEQ ID No:8                                                                    2.  invmE2                                                                   5'GGAGAAGGAGGAGGAGAG                                                          GG3'                                                                          mouse 3' PS                                                                   ×3 51                                                                            6994 SEQ                                                             ID No:9                   3.  ASmE5    5'AGAGGAGGGACAGGGCTAAG3'               mouse 3' PS                                                                   ×3 67 in E5                                                                      6889 SEQ                                                             ID No:10                  4.  S-h-     5'ATGAGGCCCCCGCAG3'                    human  3' PS                                                                  ×3 140 (Soreq                                                           1990, PNAS) 5068                                                              SEQ ID No:11                                                                         ACHE                                                                    5.  AS -                                                                     5'ACGCTTTCTTGAGGCCGC                                                          GAAGCG3'                                                                      human  loop    1119                                                           (Soreq 1990, PNAS)                                                            7969 SEQ ID No:1                                                              and                            1120L                                                                                                                                  SEQ ID                                                              No:3                      6.  AS -     5'GGCACCCTGGGCAGCCGCGAAGCG3'           human loop                                                                    1507(Soreq 1990,                                                              PNAS) 7989 SEQ ID                                                             No:2 and                  1500L            SEQ ID No:3                                                  7.  ASmE2L   5'GGGAGAGGAGGAGGAAGAGGCGCG                mouse loop    51                                                           9914 SEQ ID No:8                                                              and                                                AAGCG3'                                                                                                                  SEQ                                                           ID No:3                 __________________________________________________________________________               3' PS xn  =  last n nucleotides contain phosphorothioate            internucleotidic bonds                                                                 loop =  9 last nucleotides at the 3' are designed to form a loop     and are not part the original sequence                                                  AS =  antisense sequence                                                      S =  sense control sequence                                                   inv =  inverse (control) sequence                              

                                      TABLE 2                                     __________________________________________________________________________    Table II                                                                        series no.     delivery      oligo       no. of mice dose                                                 duration   analysis                             __________________________________________________________________________    1    i.v.                                                                              buffer                                                                              2     --  20 h.                                                                              protein assay                                                            AS-1120L    2         150 ug             RT-PCR                                                           AS-1500L    2                                           120 ug                                                                 ASmE2 PS    1         120 ug                                                               total = 7                                  2                  i.c.v.    buffer      1         50 uM      18 h.                                        protein assay                                                         ASmE2 3'L 1         200 nl                                                           histology                                                              AS-1120L    2                                                                              total = 4                                   3                  i.c.v.    buffer      2         50 uM      24 h.                                        protein assay                                                        ASmE2 PS    2         200 nl                                                  inv. mE2PS  3                                                                 ASmE2 3'L     2                                                               uninjected  1                                                                              total = 10                                   4                  i.p.      buffer      2         5 ug/gr    4 d                                          protein assay                                                       ASmE2 PS    2         body wt                                                 inv. mE2 PS 2                                                                 ASmE2 3'L   2                                                                              total = 8                                           stress+    buffer      2     5 ug/gr    4 d        protein assay                                     5                  i.p.      ASmE2 PS    2                                           body wt                                                    inv. mE2     1                                                                             total = 5                                       6                  i.c.v.    buffer      3         250 uM     48 h.                                        protein assay                                                    ASmE2 3'L   3         400 nl            RT-PCR                                AS-1120L    3                                                                              total =  9                                     __________________________________________________________________________

                                      TABLE III                                   __________________________________________________________________________    Inhibition of AChE activity by 1 μM AS-ODNs in PC12 cells.sup.a                                  B. 24 h ODN +  NGF                                                                              C. 24 h NGF, then 24 h ODN +  NGF           sp. act.                                                                             inhibition of AChE                                                                     sp. act. inhibition of AChE                                                                     sp. act. inhibition of AChE                                                             nmol/min/10.sup.3 cells                                                          activity, % nmol/min/                                                     10.sup.3 cells activity,                                                      %     nmol/min/10.sup.3                                                       cells activity, %            __________________________________________________________________________    AS1 6.7 ± 15 ± 6                                                                               7.8 ± 0.8                                                                          16 ± 7                                                                              8.5 ± 27 ± 7                      AS2           7.1 ±  0.4          11 ± 4             7.9 ± 0.3                                                               12 ± 6                                                                  12.1 ± 0.8                                                              4 ± 3                    AS3           6.1 ±   0.1          20 ± 5            8.0 ± 0.7                                                               17 ± 9                                                                  9.4 ±  0.7                                                             21 ±  8                   AS4           6.7 ±   0.6         16 ± 5             8.4 ± 0.2                                                                15 ± 10                                                                 7.5 ±  0.1                                                             36 ± 5                   AS5           7.2 ±   0.1           9 ± 5              6.5 ±                                                        0.2          28 ± 5                                                                  10.4 ± 0.1                                                             11 ± 6                AS6           7.0 ±   0.3         12 ± 5              6.9 ±                                                         0.4          23 ± 6                                                                   9.8 ±  0.7                                                             20 ± 10                                                              AS7           6.8 ±                                                        0.5         13 ± 5                                                                  7.8 ± 0.1                                                               14 ± 9                                                                  11.4 ± 0.4                                                              5 ±  3                     AS8           7.3 ±   0.1  10 ± 5             7.8 ± 0.2                                                             16 ± 8                                                                     11.4 ±  1.1       11                                                       ±  5                        I-AS5         7.4 ±   0.1           9 ± 5             7.9 ±                                                         0.7          16 ± 8                                                                  12.7 ±  1.4                                                             0 ±  1                                                               none          7.8 ±                                                       0.4             NA                                                                          9.0 ±                                                        0.4            NA                                                                    11.7 ± 0.3                                                                NA                     __________________________________________________________________________     .sup.a Averages of 6 cultures measurements and standard errors of the mea     are presented for rates of hydrolysis of acetylthiocholine  by 1,000          cells. NA  =       nonapplicable. Background due to spontaneous hydrolysi     of acetylthiocholine (7.3 nmol/min) was subtracted.                                                                              TABLE 4               

    __________________________________________________________________________    AS3 Treated                                                                       PRETREATMENT  POSTTREATMENT TREATMENT VALUE                                           Performance   Perfor     AChE Activity                              Animal      Baseline       Test  Ratio (PR1)           Baseline                                                  Test Ratio (PR2)    PR1/PR2                                                   hippocampus                              __________________________________________________________________________    2   97   142                                                                              1.46  93   84 0.90  0.62 552.30                                     4            150            147           0.98          152                                                      131  0.86          0.88                                                       125.80                                     5            100            124           1.24          140                                                      89   0.64          0.51                                                       862.00                                     6            155            153           0.99          179                                                      134  0.75          0.76                                                       122.10                                           10           150            192           1.28          221                                                     196  0.89          0.69                                                  57.20                                      Avg          130            151           1.19          157                                                      126  0.81          0.69                                                       343.88                                     S.D.         26.1           22.4          0.18          42.4                                                     40.3 0.10          0.12                                                       313.31                                   __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Control ASB                                                                       PRETREATMENT  POSTTREATMENT TREATMENT VALUE                                           Performance   Perfor     AChE Activity                              Animal      Baseline       Test  Ratio (PR1)           Baseline                                                  Test Ratio (PR2)    PR1/PR2                                                   hippocampus                              __________________________________________________________________________    1   121  133                                                                              1.10  133  162                                                                              1.22  1.11 93.90                                      3             188           182           0.97  111             122                                              1.10          1.14        89.00                                                9             143           225                                                    1.57  42              70                                                1.67          1.06        98.70                                                Avg           150            180                                                   1.21  95              118                                               1.33         1.10        93.87                                                 S.D.         27.8           37.6                                                   0.26  38.9            37.7                                              0.24          0.03         3.96          __________________________________________________________________________

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 23                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: \                                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ACGCTTTCTT GAGGC              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GGCACCCTGG GCAGC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CGCGAAGCG                - #                  - #                       - #          9                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GCCAGAGGAG GAGGAGAAGG            - #                  - #                      - # 20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TAGCGTCTAC CACCCCTGAC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - CCACGTCCTC CTGCACCGTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - ATGAACTCGA TCTCGTAGCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GGGAGAGGAG GAGGAAGAGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AGAGGAGGGA CAGGGCTAAG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TAGCATCCAA CACTCCTGAC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CTGCAATATT TTCTTGCACC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - ATGAACTCGA TTTCATAGCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GTCGTATTAT ATCCCAGCCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GTGGCTGTAA CAGTTTATTG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GACTTTGCTA TGCAT              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - GAATCGGGAC AGGGAGGAGA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "oligodeoxynucleotide"                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - TCTGTGTTAT AGCCCAGCCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: YES                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GGCCTGTAAC AGTTTATTT             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - GGAAGAGGAG GAGGAGACCG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CCCGACCCGA TATTGTGTCT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Leu Leu Ser Ala Thr Asp Thr Leu Asp Glu Al - #a Glu Arg Gln Trp Lys      1               5   - #                10  - #                15               - - Ala Glu Phe His Arg Trp Ser Ser Tyr Met Va - #l His Trp Lys Asn Gln                  20      - #            25      - #            30                   - - Phe Asp His Tyr Ser Lys Gln Asp Arg Cys Se - #r Asp Leu                          35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Leu Leu Ser Ala Thr Ala Ser Glu Ala Pro Se - #r Thr Cys Pro Gly Phe      1               5   - #                10  - #                15               - - Thr His Gly Glu Ala Ala Pro Arg Pro Gly Le - #u Pro Leu Pro Leu Leu                  20      - #            25      - #            30                   - - Leu Leu His Cys Leu Leu Leu Leu Phe Leu Se - #r His Leu Arg Arg Leu              35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Leu Leu Ser Ala Thr Gly Met Gln Gly Pro Al - #a Gly Ser Gly Trp Glu      1               5   - #                10  - #                15               - - Glu Gly Ser Gly Ser Pro Pro Gly Val Thr Pr - #o Leu Phe Ser Pro                      20      - #            25      - #            30                 __________________________________________________________________________

What is claimed is:
 1. A synthetic nuclease resistant antisenseoligodeoxynucleotide for selectively modulating humanacetylcholinesterase (AChE) production selected from the groupconsisting of5'ACGCTTTCTTGAGGC 3' SEQ ID No:1, 5'GGCACCCTGGGCAGC 3' SEQID No:2, 5'CCACGTCCTCCTGCACCGTC 3' SEQ ID No:6, 5'ATGAACTCGATCTCGTAGCC3' SEQ ID No:7.
 2. The synthetic nuclease resistant antisenseoligodeoxynucleotide as set forth in claim 1 furtherincluding5'GCCAGAGGAGGAGGAGAAGG 3' SEQ ID No:4, 5'TAGCGTCTACCACCCCTGAC3' SEQ ID No:5, 5'TCTGTGTTATAGCCCAGCCC 3' SEQ ID No:17, and5'GGCCTGTAACAGTTTATTT 3' SEQ ID No:18.
 3. A nuclease resistant antisensetargeted to a splice junction in a splice variant of human AChEmRNA. 4.The nuclease resistant antisense targeted against the splice junction asset forth in claim 3 wherein the E4-E6 junction in the E1-E4-E6 splicevariant AChEmRNA is targeted.
 5. The nuclease resistant antisensetargeted against the splice junction as set forth in claim 3 wherein theE4-E5 junction in the E1-E4-E5 splice variant AChEmRNA is targeted. 6.The nuclease resistant antisense targeted against the splice junction asset forth in claim 3 wherein the E4-I4 junction in the readthroughsplice variant AChEmRNA is targeted.
 7. A composition comprising asactive ingredient at least one synthetic nuclease resistant antisenseoligodeoxynucleotide as set forth in claim 1 in a physiologicallyacceptable carrier or diluent.
 8. A composition comprising as activeingredient at least one synthetic nuclease resistant antisenseoligodeoxynucleotide as set forth in claim 3 in a physiologicallyacceptable carrier or diluent.
 9. A synthetic nuclease resistantantisense oligodeoxynucleotide for selectively modulating humanacetylcholinesterase production in the central nervous system in a mouseselected from the group consisting of5'ACGCTTTCTTGAGGC 3' SEQ ID No:1,5'GGCACCCTGGGCAGC 3' SEQ ID No:2 5'CCACGTCCTCCTGCACCGTC 3' SEQ ID No:6,5'ATGAACTCGATCTCGTAGCC 3' SEQ ID No:7, 5'GCCAGAGGAGGAGGAGAAGG 3' SEQ IDNo:4, 5'TAGCGTCTACCACCCCTGAC 3' SEQ ID No:5, 5'TCTGTGTTATAGCCCAGCCC 3'SEQ ID No:17, and 5'GGCCTGTAACAGTTTATTT 3' SEQ ID No:18.
 10. Acomposition comprising as active ingredient at least one syntheticnuclease resistant antisense oligodeoxynucleotide as set forth in claim9 in a physiologically acceptable carrier or diluent.
 11. A method torestore balanced cholinergic signaling in the brain in mice in need ofsuch treatment comprising administering to a mouse in need of suchtreatment a therapeutically effective amount of at least one of asynthetic nuclease resistant antisense oligodeoxynucleotide according toclaim
 1. 12. The synthetic nuclease resistant antisenseoligodeoxynucleotides as set forth in claim 1 wherein the nucleaseresistance is selected from the group consisting of havingphosphorothioate bonds linking between the four 3'-terminus nucleotidebases, having a 9 nucleotide loop forming sequence at the 3'-terminushaving the nucleotide sequence CGCGAAGCG (SEQ ID No:3), at least onenucleotide is O-methylated, and at least one nucleotide is fluoridated.13. The synthetic nuclease resistant antisense oligodeoxynucleotides asset forth in claim 3 wherein the nuclease resistance is selected fromthe group consisting of having phosphorothioate bonds linking betweenthe four 3'-terminus nucleotide bases, having a 9 nucleotide loopforming sequence at the 3'-terminus having the nucleotide sequenceCGCGAAGCG (SEQ ID No:3), at least one nucleotide is O-methylated, and atleast one nucleotide is fluoridated.
 14. A method of determining theefficacy of a synthetic nuclease resistant antisenseoligodeoxynucleotide by screening in a transgenic harboring theintegrated human AChE gene and in cortico-hippocampal brain slices froma transgenic mouse harboring the integrated human ACHE gene whereby theefficacy of the synthetic nuclease resistant antisenseoligodeoxynucleotide to selectively modulate human acetylcholinesteraseproduction can be determined.